Application of compound or traditional chinese medicine extract in preparation of nucleic acid delivery agent and related products thereof

ABSTRACT

The present application relates to extracting a plurality of compounds from a traditional Chinese medicine, or synthesizing a compound capable of promoting nucleic acid delivery, and utilizing the extracted compound, or a plurality of combinations to promote absorption and entry of a nucleic acid such as sRNA into a target cell, and to facilitate the entry of a nucleic acid into a target site in a subject in need thereof.

SEQUENCE LISTING

A copy of the Sequence Listing is submitted with the specificationelectronically via EFS-Web as an ASCII formatted sequence listing with afile name of “074844-8002WO01-SL-20190910 ST25.txt”, a creation date ofSep. 23, 2019, and a size of 6,366 bytes. The sequence listing containedin this ASCII formatted document is part of the specification and isherein incorporated by reference in its entirety.

TECHNICAL FIELD

The present application relates to various compounds that are extractedfrom traditional Chinese medicines or are synthetic and that are capableof promoting nucleic acid delivery, and use of the extracted compoundsor various combinations thereof to promote the absorption and entry ofnucleic acids, such as sRNA, into target cells, and to promote entryinto target sites in vivo in a subject in need thereof.

BACKGROUND

In the past few decades, the concept of using nucleic acid molecules,including RNA molecules, as therapeutic drugs has moved from concept toclinical reality. In fact, nucleic acid molecules have many propertiesthat make it a therapeutic drug. They can fold to form complexconformations that allow them to bind to proteins, small molecules orother nucleic acids, and some can even form catalytic centers. Smallinterfering RNA (siRNA), as an effector molecule of RNAi, has anincreasingly broad prospect as a therapeutic drug. At present, a varietyof siRNA drugs have entered clinical trials, indicating a gooddevelopment prospect. Generally, siRNA, miRNA and other non-coding smallRNA are indiscriminately referred to as small nucleic acids or small RNA(sRNA). However, since nucleic acid molecules are easily degraded andhave a relatively short half-life in vivo, they are generally consideredto be a poor choice as therapeutic drugs.

Therefore, how to efficiently deliver nucleic acid molecules, includingsmall RNA, to a target organ and a target cell in vivo in order toachieve their biological activity and therapeutic or preventive effectsis a problem to be considered by those skilled in the art.

SUMMARY OF INVENTION

After extensive tests, the inventor has unexpectedly discovered somelipid components in some traditional Chinese medicines (includingRhodiola crenulata, Taraxacum mongolicum, Andrographis paniculata andLonicera japonica), and these lipids derived from the traditionalChinese medicines can promote absorption/entry of nucleic acids, such assmall RNA into cells and/or target parts in a subject in need thereof.In the present invention, the lipid component is synthetic.

Specifically, in one aspect, the present application relates to acompound having the following structure extracted from a traditionalChinese medicine, and use of the compound for the manufacture of areagent for nucleic acid delivery:

wherein, L₁, L₂ or L₃ is absent, or L₁, L₂ and L₃ are each independentlyselected from the group consisting of —C(O)O—CH₂—, —CH(OH)—,—C(O)—NH—CH₂—, —CH₂—O—C(O)—, —CH₂—NH—C(O)—, —C(O)O—, —C(O)NH—, —OC(O)—,—NH—C(O)—, —CH₂—,

with the proviso that at most two of L₁, L₂, and L₃ are absent;

with respect to the divalent groups L₁, L₂, the dash “-” on the leftside is linked to the groups A and B, respectively, and the dash “-” onthe right side is linked to the central carbon atom;

with respect to the divalent group L₃, the dash “-” on the left side islinked to the central carbon atom, and the dash “-” on the right side islinked to the group Q;

A, B and Q are each independently selected from the group consisting ofH, —OH, C₁₋₂₀ alkyl, C₁₋₂₀ alkenyl, C₁₋₂₀ heteroalkyl, C₁₋₂₀heteroalkenyl, —NH₂, and —NR₃ ⁺, R is H or C₁₋₆ alkyl; and

n is an integer 0, 1, 2, 3 or 4;

In one embodiment, in said use, in the structure of the compound:

L₁ is absent, or L₁ is selected from —C(O)O—CH₂— and —CH(OH)—,

L₂ is absent, or L₂ is selected from —C(O)O— and —C(O)NH—,

L₃ is absent, or L₃ is selected from —C(O)O—, —CH₂—OC(O)—, —CH₂— and

A is selected from the group consisting of H, C₁₋₂₀ alkyl and C₁₋₂₀alkenyl;

B is selected from the group consisting of H, —NH₂, C₁₋₂₀ alkyl andC₁₋₂₀ alkenyl;

Q is selected from the group consisting of H, —OH, C₁₋₂₀ alkyl and C₁₋₂₀alkenyl, and —NR₃ ⁺, wherein R is H or C₁₋₆ alkyl.

In one embodiment, the compound has the following formula:

In one embodiment, in the structure of the compound:

A is selected from the group consisting of H, C₁₀₋₂₀ alkyl and C₁₀₋₂₀alkenyl;

B is selected from the group consisting of H, —NH₂, C₁₀₋₂₀ alkyl andC₁₀₋₂₀ alkenyl;

Q is selected from the group consisting of H, —OH, C₁₀₋₂₀ alkyl andC₁₀₋₂₀ alkenyl, and —NR₃ ⁺, wherein R is H or C₁₄ alkyl.

In one embodiment, in the structure of the compound:

A is selected from the group consisting of H, a straight-chain C₁₅₋₁₈alkyl group and a straight-chain C₁₅₋₁₈ alkenyl group;

B is selected from the group consisting of H, —NH₂, a straight-chainC₁₅₋₁₈ alkyl group and a straight-chain C₁₅₋₁₈ alkenyl group;

Q is selected from the group consisting of H, —OH, a straight-chainC₁₅₋₁₈ alkyl group and a straight-chain C₁₅₋₁₈ alkenyl group, and —NR₃ ⁺wherein R is H or a C₁₄ alkyl group; and the alkenyl group in the A, B,Q has 1-5 double bonds.

In one embodiment, in the A, B, Q of the structure, the alkenyl grouphas 1-3 double bonds and is in a Z configuration.

In one embodiment, the said compound is selected from the followingformulas:

wherein

A is selected from a straight-chain C₁₅₋₁₈ alkyl group and astraight-chain C₁₅₋₁₈ alkenyl group;

B is selected from a straight-chain C₁₅₋₁₈ alkyl group and astraight-chain C₁₅₋₁₈ alkenyl group;

Q is selected from the group consisting of H, —OH, a straight-chainC₁₅₋₁₈ alkyl group and a straight-chain C₁₅₋₁₈ alkenyl group, and —NR₃ ⁺wherein R is H or methyl; and

L₃ is —C(O)O—.

In one embodiment, the compound is lysolecithin, ceramide, diglyceride,phosphatidylethanolamine, phosphatidylcholine, triglyceride,monogalactosyl diglyceride, sphingosine, phosphatidyl ethanol,monoacylglycerol, fatty acid, platelet activating factor, or dimethylphosphatidyl ethanolamine.

In one embodiment, the compound is a lipid shown in Table 1.

In one embodiment, the compound is a lipid shown in Table 1 as No. 11,No. 12, No. 41, No. 71, No. 38, No. 64, No. 40, No. 37, No. 39, No. 60or No. 62.

In a second aspect, the present application relates to use of acombination comprising any one or more of the above compounds.Preferably any one or more of the lipids selected Table 1, for themanufacture of a nucleic acid delivery reagent. Preferably, thecombination comprises any one of the lipids in Table 1 as No. 11, No.12, No. 41, No. 71, No. 38, No. 64, No. 40, No. 37, No. 39, No. 60 orNo. 62, or combination thereof with any one or more of the other lipidsin Table 1.

In a third aspect, the present application relates to use of atraditional Chinese medicine for the manufacture of a nucleic aciddelivery reagent.

In one embodiment, the traditional Chinese medicine is selected fromRhodiola crenulata, Taraxacum mongolicum, Andrographis paniculata andLonicera japonica Chinese medicine decoction pieces.

In one embodiment, the reagent comprises a compound extracted from atraditional Chinese medicine. Preferably, the reagent comprises any oneor more of the above compounds, preferably any one or more lipidsselected from Table 1. Preferably, the reagent comprises any one of thelipids shown in Table 1 as No. 11, No. 12, No. 41, No. 71, No. 38, No.64, No. 40, No. 37, No. 39, No. 60 or No. 62, or its combination withany one or more of the other lipids shown in Table 1.

In one embodiment, the compound is extracted by decoction of atraditional Chinese medicine. In another embodiment, the compound isextracted by soaking the traditional Chinese medicine pieces in water,followed by performing intense heating and slow heating sequentially,and then the heated Chinese medicine soup is concentrated, and then issequentially added with chloroform-methanol, chloroform and water forstirring, and the chloroform layer is obtained.

In one embodiment, the compound has the structure shown in any one ofthe preceding embodiments.

In one embodiment, the compound is selected from lysolecithin, ceramide,diglyceride, phosphatidylethanolamine, phosphatidylcholine,triglyceride, monogalactosyldiglyceride, (neural) sphingosine,phosphatidyl ethanol, monoacylglycerol, fatty acid, platelet activatingfactor, or dimethyl phosphatidyl ethanolamine.

In one embodiment, wherein the compound is selected from Table 1.

In one embodiment, wherein the compound is the lipid shown in Table 1 asNo. 11, No. 12, No. 41, No. 71, No. 38, No. 64, No. 40, No. 37, No. 39,No. 60 or No. 62.

In one embodiment, wherein the delivery comprises in vitro celldelivery, or in vivo gastrointestinal delivery.

In one embodiment, the use includes the manufacture of lipid nucleicacid mixture.

In one embodiment, the lipid nucleic acid mixture is manufactured by aboiling method, or by a reverse evaporation method, or by direct mixing.

In one embodiment, temperature in said boiling method is from about 25°C. to about 100° C., preferably from about 80° C. to about 100° C.;temperature in the reverse evaporation method is from about 25° C. toabout 70° C., preferably about 55° C.

In a fourth aspect, the present application relates to a pharmaceuticalcomposition comprising a compound of the structure of any one of thepreceding embodiments and a nucleic acid. Preferably, the saidpharmaceutical composition comprises any one or more of the abovecompounds, preferably one or more lipids selected from Table 1.

Preferably, the pharmaceutical composition comprises any one of thelipids shown in Table 1 as No. 11, No. 12, No. 41, No. 71, No. 38, No.64, No. 40, No. 37, No. 39, No. 60 or No. 62, or its combination withany one or more of the other lipids shown in Table 1, or its combinationwith any one or more lipids and other related chemicals.

In one embodiment, in said pharmaceutical composition, the lipid andnucleic acid are at least partially or wholly existed in the form oflipid nucleic acid mixture.

In one embodiment, in said pharmaceutical composition, the lipid nucleicacid mixture is manufactured by a boiling method, or by a reverseevaporation method, or by direct mixing.

In one embodiment, in said pharmaceutical composition, temperature inthe boiling method is from 25° C. to about 100° C., preferably fromabout 80° C. to 100° C. temperature in the reverse evaporation method isfrom about 25° C. to about 70° C., preferably about 55° C.

In a fifth aspect, the present application relates to a kit comprisingthe lipid and the nucleic acid of the preceding embodiments, wherein thelipid and nucleic acid are each independently provided in a firstcontainer and a second container, the first container and the secondcontainer are the same or different. Preferably, the kit comprises anyone or more of the above compounds, preferably any one or more lipidsselected from Table 1. Preferably, the kit comprises any one of thelipids shown in Table 1 as No. 11, No. 12, No. 41, No. 71, No. 38, No.64, No. 40, No. 37, No. 39, No. 60, or No. 62, or its combination withany one or more of the other lipids shown in Table 1, or its combinationwith any one or more lipids and other related chemicals.

In one embodiment, in said pharmaceutical composition, the lipid andnucleic acid are at least partially or wholly manufactured into lipidnucleic acid mixture immediately prior to use.

In one embodiment, in said pharmaceutical composition, the lipid nucleicacid mixture is manufactured by a boiling method, or by a reverseevaporation method, or by direct mixing.

In one embodiment, in said pharmaceutical composition, temperature inthe boiling method is from 25° C. to about 100° C., preferably about100° C., temperature in the reverse evaporation method is from about 25°C. to about 70° C., preferably about 55° C.

In a sixth aspect, the present application relates to a method ofdelivering a nucleic acid into a target cell, comprising providing thenucleic acid in a form of the pharmaceutical composition or the kit ofany one of the preceding embodiments.

In a seventh aspect, the present application relates to a method ofdelivering a nucleic acid into a subject in vivo in need thereof,comprising providing the nucleic acid in a form of the pharmaceuticalcomposition or the kit of any one of the preceding.

In one embodiment, in the above method, the subject is a human or ananimal, such as a mammal.

In one embodiment, in the above method, the nucleic acid is delivered toblood circulation or a target tissue/cell in a subject in vivo.

In one embodiment, the above method comprises directly delivering thepharmaceutical composition or the kit of any one of the precedingembodiments to a subject in need thereof by digestive tract.

In any one of the preceding aspects or embodiments, for example in thepharmaceutical composition or the kit, the nucleic acid and the lipidare manufactured for topical administration and/or injection.

In any one of the preceding aspects or embodiments, for example in thepharmaceutical composition or the kit, wherein the nucleic acid and thelipid are manufactured for digestive administration, respiratoryadministration and/or injection.

In any one of the preceding aspects or embodiments, for example in thepharmaceutical composition or the kit, wherein the nucleic acid and thelipid are manufactured for oral administration, inhalationadministration and/or injection.

In any one of the preceding aspects or embodiments, for example in thepharmaceutical composition or the kit, wherein the nucleic acid is asmall RNA.

In any one of the preceding aspects or embodiments, for example in thepharmaceutical composition or the kit, wherein the nucleic acid has astem-loop structure.

In any one of the preceding aspects or embodiments, for example in thepharmaceutical composition or the kit, wherein the small RNA has alength of 14-32 bp or 18-24 bp, for example, a length of 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32 bp.

In any one of the preceding aspects or embodiments, the pharmaceuticalcomposition, the kit or the compound can be orally administered.

In any one of the preceding aspects or embodiments, the nucleic acid canbe used for treating a disease, such as cancer, for example gastriccancer or lung cancer.

In any one of the preceding aspects or embodiments, a lipid combinationcan be used, and the lipid combination is any one of the following: alipid combination of No. 8:No. 41=6:1; a lipid combination of No. 38:No.41=6:1; a lipid combination of No. 39:No. 41=6:1; a lipid combination ofNo. 40:No. 41=6:1; a lipid combination of No. 38:No. 12:No. 41:No.29=1:1:2:1; a lipid combination of No. 40:No. 12:No. 41=2:4:3; a lipidcombination of No. 12:No. 41=1:6; a lipid combination of No. 12:No.41=1:1; a lipid combination of No. 12:No. 41=6:1; a lipid combination ofNo. 40:No. 12:No. 41=2:2:2; a lipid combination of No. 4:No. 12:No.41=1:1:1; DG combination of No. 1:No. 2:No. 3:No. 19:No. 35=1:1:1:1:1;TG combination of No. 6:No. 9:No. 10:No. 13:No. 15:No. 16:No. 18:No.20:No. 21:No. 22:No. 23:No. 24:No. 25:No. 26:No. 27:No. 28:No. 32:No.33=1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1; LPC combination of No. 36:No.37=1:1; PC combination of No. 11:No. 12=1:1; PE combination of No. 8:No.38=1:1; Cer combination of No. 4:No. 14=1:1; So combination of No.17:No. 30:No. 31=1:1:1; an equal volume combination of No. 1-36 withoutNo. 5, No. 7; an equal volume combination of No. 1-36 without No. 5, No.7, No. 34; an equal volume combination of No. 1-36 without No. 5, No. 7,No. 1, No. 2, No. 3, No. 19, No. 35; an equal volume combination of No.1-36 No. 5, No. 7, No. 6, No. 9, No. 10, No. 13, No. 15, No. 16, No. 18,No. 20, No. 21, No. 22, No. 23, No. 24, No. 25, No. 26, No. 27, No. 28,No. 32, No. 33; an equal volume combination of No. 1-36 without No. 5,No. 7, No. 36, No. 37; an equal volume combination of No. 1-36 withoutNo. 5, No. 7, No. 11, No. 12; an equal volume combination of No. 1-36without No. 5, No. 7, No. 8 in; an equal volume combination of No. 1-36without No. 5, No. 7, No. 4, No. 14; an equal volume combination of No.1-36 without No. 5, No. 7, No. 29; a lipid combination of No. 1:No.34=2:1; a lipid combination of No. 1: said DG composition=2:1; a lipidcombination of No. 1: said TG composition=2:1; a lipid combination ofNo. 1: said LPC composition=2:1; a lipid combination of No. 1:No. 8=2:1;a lipid combination of No. 1:No. 12=2:1; a lipid combination of No. 1:said Cer composition=2:1; a lipid combination of No. 1: said Socomposition=2:1; a lipid combination of No. 1:No. 29=2:1; a lipidcombination of No. 1:No. 8:No. 12=1:1:1; a lipid combination of No.8:No. 34=2:1; a lipid combination of No. 8: said DG composition=2:1; alipid combination of No. 8: said TG composition=2:1; a lipid combinationof No. 8: said LPC composition=2:1; a lipid combination of No. 8:No.37=4:1; a lipid combination of No. 8:No. 12=2:1; a lipid combination ofNo. 8: said Cer composition=2:1; a lipid combination of No. 8: said Socomposition=2:1; a lipid combination of No. 8:No. 31=6:1; a lipidcombination of No. 8:No. 29=2:1; a lipid combination of No. 12:No.34=2:1; a lipid combination of No. 12: said DG composition=2:1; a lipidcombination of No. 12: said TG composition=2:1; a lipid combination ofNo. 12: said LPC composition=2:1; a lipid combination of No. 12:No.8=2:1; a lipid combination of No. 12: said Cer composition=2:1; a lipidcombination of No. 12: said So composition=2:1; a lipid combination ofNo. 12:No. 29=2:1; a lipid combination of No. 12:No. 8:No. 1&2=2:1:1; alipid combination of No. 12:No. 8:No. 15=2:1:1; a lipid combination ofNo. 12:No. 8:No. 36&37=2:1:1; a lipid combination of No. 12:No. 8:No.11=2:1:1; a lipid combination of No. 12:No. 8:No. 12=2:1:1; a lipidcombination of No. 12:No. 8:No. 4=2:1:1; a lipid combination of No.12:No. 8:No. 31=2:1:1; a lipid combination of No. 12:No. 8:No. 29=2:1:1;a lipid combination of No. 12:No. 8:No. 34=3:2:1; a lipid combination ofNo. 12:No. 8:No. 34=4:2:3; a lipid combination of No. 12:No. 8:No.2=4:2:3; a lipid combination of No. 12:No. 8:No. 2=16:8:3; a lipidcombination of No. 12:No. 8:No. 32=4:2:3; a lipid combination of No.12:No. 8:No. 37=4:2:3; a lipid combination of No. 12:No. 8:No. 11=4:2:3;a lipid combination of No. 12:No. 8:No. 38=4:2:3; a lipid combination ofNo. 12:No. 8:No. 4=4:2:3; a lipid combination of No. 12:No. 8:No.31=4:2:3; a lipid combination of No. 12:No. 8:No. 29=4:2:3; a lipidcombination of No. 12:No. 8:No. 29:No. 31=2:1:1:1; a lipid combinationof No. 12:No. 8:No. 29:No. 31:No. 34=4:2:2:2:5; a lipid combination ofNo. 12:No. 8:No. 29:No. 31:No. 2=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 32=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 11=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 37=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 38=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 4=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 4:No. 1:No. 16=2:1:1:3:2:2:3; a lipidcombination of No. 1:No. 8:No. 12:No. 1&2=2:2:2:3; a lipid combinationof No. 1:No. 8:No. 12:No. 15=2:2:2:3; a lipid combination of No. 1:No.8:No. 12:No. 36&37=2:2:2:3; a lipid combination of No. 1:No. 8:No.12:No. 12=2:2:2:3; a lipid combination of No. 1:No. 8:No. 12:No.4=2:2:2:3; a lipid combination of No. 1:No. 8:No. 12:No. 31=2:2:2:3; alipid combination of No. 1:No. 8:No. 12:No. 29=2:2:2:3; a lipidcombination of No. 8:No. 34:No. 1&2=2:1:1; a lipid combination of No.8:No. 34:No. 15=2:1:1; a lipid combination of No. 8:No. 34:No.36&37=2:1:1; a lipid combination of No. 8:No. 34:No. 12=2:1:1; a lipidcombination of No. 8:No. 34:No. 4=2:1:1; a lipid combination of No.8:No. 34:No. 31=2:1:1; a lipid combination of No. 8:No. 34:No. 29=2:1:1;a lipid combination of No. 8:No. 31:No. 34=12:3:5; a lipid combinationof No. 8:No. 31:No. 2=12:3:5; a lipid combination of No. 8:No. 31:No.37=12:3:5; a lipid combination of No. 8:No. 31:No. 11=12:3:5; a lipidcombination of No. 8:No. 31:No. 12=12:3:5; a lipid combination of No.8:No. 31:No. 4=12:3:5; a lipid combination of No. 8:No. 31:No.29=12:3:5; a lipid combination of No. 8:No. 31:No. 32=12:3:5; a lipidcombination of No. 8:No. 4:No. 34=12:3:5; a lipid combination of No.8:No. 4:No. 2=12:3:5; a lipid combination of No 0.8:No. 4:No. 37=12:3:5;a lipid combination of No. 8:No. 4:No. 12=12:3:5; a lipid combination ofNo. 8:No. 4:No. 31=12:3:5; a lipid combination of No. 8:No. 4:No.29=12:3:5; a lipid combination of No. 8:No. 4:No. 32=12:3:5; a lipidcombination of No. 38:No. 34=2:1; a lipid combination of No. 38:No.1=2:1; a lipid combination of No. 38:No. 2=2:1; a lipid combination ofNo. 38:No. 1&2=2:1; a lipid combination of No. 38:No. 15=2:1; a lipidcombination of No. 38:No. 32=2:1; a lipid combination of No. 38:No.37=2:1; a lipid combination of No. 38:No. 37=4:1; a lipid combination ofNo. 38:No. 11=2:1; a lipid combination of No. 38:No. 12=2:1; a lipidcombination of No. 38:No. 11&12=2:1; a lipid combination of No. 38:No.12=4:1; a lipid combination of No. 38:No. 8=2:1; a lipid combination ofNo. 38:No. 4=2:1; a lipid combination of No. 38: So (30)=2:1; a lipidcombination of No. 38:No. 31=2:1; a lipid combination of No. 38:No.29=2:1; a lipid combination of No. 1:No. 38:No. 12:No. 34=2:2:2:3; alipid combination of No. 1:No. 38:No. 12:No. 15=2:2:2:3; a lipidcombination of No. 1:No. 38:No. 12:No. 37=2:2:2:3; a lipid combinationof No. 1:No. 38:No. 12:No. 8=2:2:2:3; a lipid combination of No. 1:No.38:No. 12:No. 4=2:2:2:3; a lipid combination of No. 1:No. 38:No. 12:No.31=2:2:2:3; a lipid combination of No. 1:No. 38:No. 12:No. 29=2:2:2:3; alipid combination of No. 38:No. 34:No. 1=2:1:3; a lipid combination ofNo. 38:No. 34:No. 15=2:1:3; a lipid combination of No. 38:No. 34:No.37=2:1:3; a lipid combination of No. 38:No. 34:No. 12=2:1:3; a lipidcombination of No. 38:No. 34:No. 8=2:1:3; a lipid combination of No.38:No. 34:No. 4=2:1:3; a lipid combination of No. 38:No. 34:No.31=2:1:3; a lipid combination of No. 38:No. 34:No. 29=2:1:3; a lipidcombination of No. 38:No. 12:No. 1=2:1:3; a lipid combination of No.38:No. 12:No. 2=4:1:3; a lipid combination of No. 38:No. 12:No.15=2:1:3; a lipid combination of No. 38:No. 12:No. 37=2:1:3; a lipidcombination of No. 38:No. 12:No. 8=2:1:3; a lipid combination of No.38:No. 12:No. 4=2:1:3; a lipid combination of No. 38:No. 12:No.31=2:1:3; a lipid combination of No. 38:No. 12:No. 29=2:1:3; a lipidcombination of No. 38:No. 12:No. 1:No. 15:No. 34=22:22:22:33:36; a lipidcombination of No. 38:No. 12:No. 1:No. 15:No. 37=22:22:22:33:36; a lipidcombination of No. 38:No. 12:No. 1:No. 15:No. 4=22:22:22:33:36; a lipidcombination of No. 38:No. 12:No. 1:No. 15:No. 31=22:22:22:33:36; a lipidcombination of No. 38:No. 12:No. 1:No. 15:No. 29=22:22:22:33:36; a lipidcombination of No. 38:No. 34:No. 37:No. 1=44:22:33:36; a lipidcombination of No. 38:No. 34:No. 37:No. 15=44:22:33:36; a lipidcombination of No. 38:No. 34:No. 37:No. 12=44:22:33:36; a lipidcombination of No. 38:No. 34:No. 37:No. 4=44:22:33:36; a lipidcombination of No. 38:No. 34:No. 37:No. 31=44:22:33:36; a lipidcombination of No. 38:No. 12:No. 4:No. 34=44:22:33:36; a lipidcombination of No. 38:No. 12:No. 4:No. 1=44:22:33:36; a lipidcombination of No. 38:No. 12:No. 4:No. 15=44:22:33:36; a lipidcombination of No. 38:No. 12:No. 4:No. 37=44:22:33:36; a lipidcombination of No. 38:No. 12:No. 4:No. 37=8:2:5:3; a lipid combinationof No. 38:No. 12:No. 4:No. 31=44:22:33:36; a lipid combination of No.38:No. 12:No. 4:No. 29=44:22:33:36; a lipid combination of No. 38:No.12:No. 4:No. 29:No. 34=88:44:66:72:135; a lipid combination of No.38:No. 12:No. 4:No. 29:No. 1=88:44:66:72:135; a lipid combination of No.38:No. 12:No. 4:No. 29:No. 15=88:44:66:72:135; a lipid combination ofNo. 38:No. 12:No. 4:No. 29:No. 37=88:44:66:72:135; a lipid combinationof No. 38:No. 12:No. 4:No. 29:No. 31=88:44:66:72:135; a lipidcombination of No. 38:No. 12:No. 4:No. 2=20:10:15:9; a lipid combinationof No. 38:No. 12:No. 4:No. 6=20:10:15:9; a lipid combination of No.38:No. 12:No. 4:No. 17=20:10:15:9; a lipid combination of No. 38:No.12:No. 4:No. 29=20:10:15:9; a lipid combination of No. 38:No. 12:No.4:No. 34=20:10:15:9; a lipid combination of No. 38:No. 12:No. 4:No.37=20:10:15:9; a lipid combination of No. 38:No. 12:No. 31:No.34=2:1:3:3; a lipid combination of No. 38:No. 12:No. 31:No. 1=2:1:3:3; alipid combination of No. 38:No. 12:No. 31:No. 15=2:1:3:3; a lipidcombination of No. 38:No. 12:No. 31:No. 37=2:1:3:3; a lipid combinationof No. 38:No. 12:No. 31:No. 4=2:1:3:3; a lipid combination of No. 38:No.12:No. 31:No. 29=2:1:3:3; a lipid combination of No. 38:No. 34:No.37:No. 31:No. 1=88:44:66:72:135; a lipid combination of No. 38:No.34:No. 37:No. 31:No. 15=88:44:66:72:135; a lipid combination of No.38:No. 34:No. 37:No. 31:No. 12=88:44:66:72:135; a lipid combination ofNo. 38:No. 34:No. 37:No. 31:No. 4=88:44:66:72:135; a lipid combinationof No. 38:No. 34:No. 37:No. 31:No. 29=88:44:66:72:135; a lipidcombination of No. 38:No. 37:No. 34=4:2:3; a lipid combination of No.38:No. 37:No. 1=4:2:3; a lipid combination of No. 38:No. 37:No. 2=4:2:3;a lipid combination of No. 38:No. 37:No. 1&2=4:2:3; a lipid combinationof No. 38:No. 37:No. 2=32:8:5; a lipid combination of No. 38:No. 37:No.32=32:8:5; a lipid combination of No. 38:No. 37:No. 15=4:2:3; a lipidcombination of No. 38:No. 37:No. 32=4:2:3; a lipid combination of No.38:No. 37:No. 8=4:2:3; a lipid combination of No. 38:No. 37:No.11=4:2:3; a lipid combination of No. 38:No. 37:No. 12=4:2:3; a lipidcombination of No. 38:No. 37:No. 11&12=4:2:3; a lipid combination of No.38:No. 37:No. 12=4:1:1; a lipid combination of No. 38:No. 37:No.4=4:2:3; a lipid combination of No. 38:No. 37:No. 30=4:2:3; a lipidcombination of No. 38:No. 37:No. 31=4:2:3; a lipid combination of No.38:No. 37:No. 29=4:2:3; a lipid combination of No. 8:No. 37:No.32=4:1:2; a lipid combination of No. 8:No. 37:No. 2=4:1:2; a lipidcombination of No. 38:No. 37:No. 15:No. 34=64:16:10:45; a lipidcombination of No. 38:No. 37:No. 15:No. 1=64:16:10:45; a lipidcombination of No. 38:No. 37:No. 15:No. 12=64:16:10:45; a lipidcombination of No. 38:No. 37:No. 15:No. 4=64:16:10:45; a lipidcombination of No. 38:No. 37:No. 15:No. 31=64:16:10:45; a lipidcombination of No. 38:No. 37:No. 15:No. 29=64:16:10:45; a lipidcombination of No. 38:No. 2:No. 37=4:2:3; a lipid combination of No.38:No. 2:No. 31=4:2:3; a lipid combination of No. 38:No. 2:No. 29=4:2:3;a lipid combination of No. 38:No. 2:No. 34=4:2:3; a lipid combination ofNo. 38:No. 2:No. 32=4:2:3; a lipid combination of No. 38:No. 2:No.12=4:2:3; a lipid combination of No. 38:No. 2:No. 12=4:5:1; a lipidcombination of No. 38:No. 2:No. 4=4:2:3. In one embodiment, lipids No.1&2, No. 11&12 or No. 36&37 can represent lipids No. 1 and No. 2 in anyratio, lipids No. 11 or No. 12 in any ratio, lipids No. 36 and No. 37 inany ratio, respectively.

The present application also provides a compound having a structure ofthe following formula, a combination or a composition comprising thecompound, and a method of using the compound, combination or compositionfor nucleic acid delivery, and use of the compound, combination orcomposition for the manufacture of a nucleic acid delivery reagent:

wherein

L₁, L₂ or L₃ is absent, or L₁, L₂ and L₃ are each independently selectedfrom the group consisting of —C(O)O—CH₂—, —CH(OH)—, —CH₂—OC(O), —C(O)O—,—C(O)NH—;

with the proviso that at most two of L₁, L₂ and L₃ are absent;

with respect to the divalent groups L₁, L₂, the dash “-” on the leftside is linked to the groups A and B, respectively, and the dash “-” onthe right side is linked to the central carbon atom;

with respect to the divalent group L₃, the dash “-” on the left side islinked to the central carbon atom, and the dash “-” on the right side islinked to the group Q;

A, B and Q is independently selected from the group consisting of H,—OH, C₁₋₂₀ alkyl, C₁₋₂₀ alkenyl, —NH₂, and —NR₃ ⁺, R is H or C₁₋₆ alkyl.

In one embodiment, the compound can have the following structure:

wherein

A is selected from the group consisting of a straight-chain C₁₀₋₂₀ alkylgroup and a straight-chain C₁₀₋₂₀ alkenyl group;

B is selected from the group consisting of a straight-chain C₁₀₋₂₀ alkylgroup and a straight-chain C₁₀₋₂₀ alkenyl group;

Q is —OH;

preferably,

A is selected from the group consisting of a straight-chain C₁₅₋₂₀ alkylgroup and a straight-chain C₁₅₋₂₀ alkenyl group;

B is selected from the group consisting of a straight-chain C₁₅₋₂₀ alkylgroup and a straight-chain C₁₅₋₂₀ alkenyl group;

Q is —OH;

preferably,

A is selected from the group consisting of a straight-chain C₁₅₋₁₈ alkylgroup and a straight-chain C₁₅₋₁₈ alkenyl group;

B is selected from the group consisting of a straight-chain C₁₅₋₁₈ alkylgroup and a straight-chain C₁₅₋₁₈ alkenyl group;

Q is —OH.

In another embodiment, the said compound can have the followingstructure:

wherein

A is selected from the group consisting of a straight-chain C₁₀₋₂₀ alkylgroup and a straight-chain C₁₀₋₂₂ alkenyl group;

B is selected from the group consisting of a straight-chain C₁₀₋₂₀ alkylgroup and a straight-chain C₁₀₋₂₂ alkenyl group;

Q is selected from the group consisting of a straight-chain C₁₀₋₂₀ alkylgroup and a straight-chain C₁₀₋₂₂ alkenyl group;

preferably,

A is selected from the group consisting of a straight-chain C₁₅₋₁₈ alkylgroup and a straight-chain C₁₅₋₂₂ alkenyl group;

B is selected from the group consisting of a straight-chain C₁₅₋₁₈ alkylgroup and a straight-chain C₁₅₋₂₂ alkenyl group;

Q is selected from the group consisting of a straight-chain C₁₅₋₁₈ alkylgroup and a straight-chain C₁₅₋₂₂ alkenyl group;

preferably,

A is selected from the group consisting of a straight-chain C₁₅₋₁₈ alkylgroup and a straight-chain C₁₅₋₂₀ alkenyl group;

B is selected from the group consisting of a straight-chain C₁₅₋₁₈ alkylgroup and a straight-chain C₁₅₋₂₀ alkenyl group;

Q is selected from the group consisting of a straight-chain C₁₅₋₁₈ alkylgroup and a straight-chain C₁₅₋₂₀ alkenyl group.

In another embodiment, the compound can have the following structure:

wherein

A is selected from the group consisting of a straight-chain C₁₀₋₂₀ alkylgroup and a straight-chain C₁₀₋₂₀ alkenyl group;

B is selected from the group consisting of a straight-chain C₁₀₋₂₀ alkylgroup and a straight-chain C₁₀₋₂₀ alkenyl group;

Q is —OH;

preferably,

A is selected from the group consisting of a straight-chain C₁₅₋₂₀ alkylgroup and a straight-chain C₁₅₋₁₈ alkenyl group;

B is selected from the group consisting of a straight-chain C₁₅₋₁₈ alkylgroup and a straight-chain C₁₅₋₁₈ alkenyl group;

Q is —OH;

preferably,

A is a straight-chain C₁₅₋₂₀ alkyl group;

B is a straight-chain C₁₅₋₁₈ alkyl group;

Q is —OH.

In another embodiment, the compound can have the following structure:

wherein

A is selected from the group consisting of a straight-chain C₁₀₋₂₀ alkylgroup and a straight-chain C₁₀₋₂₀ alkenyl group;

Q is —OH;

preferably,

A is selected from the group consisting of a straight-chain C₁₀₋₂₀ alkylgroup and a straight-chain C₁₅₋₁₈ alkenyl group;

Q is —OH;

preferably,

A is a straight-chain C₁₅₋₂₀ alkyl group;

Q is —OH.

In any aspects or embodiments of the present application, the compoundcan be a compound as described above.

In any aspects or embodiments of the present application, the compound,the extract or the composition can be derived synthetically, naturallyor extracted from a traditional Chinese medicine.

The above technical solutions provided by the present application cansignificantly improve the high-efficiency targeted delivery of a nucleicacid, and overcome the shortcomings in the prior art of nucleic acidliposome, including low encapsulation rate, poor safety, poor stability,complicated manufacture process, heterogeneity in product, lowreproducibility, and the to-be-improved targeting.

TABLE 1-1 List of 69 lipids derived from traditional Chinese medicineWorking Catalogue Concentration No. Manufacturer # Abbreviation (mg/mL)1 Avanti 110882 DG(18:0/18:0/0:0) 5 2 Avanti 110883 DG(18:0/16:0/0:0) 53 Avanti 800816C DG(16:0/16:0/0:0) 10 4 Avanti 860627P C18Dihydroceramide 10 (d18:0/18:0) 6 Avanti 110613 TG(18:1/18:1/18:1) 1 8Avanti 850756C PE(16:0/18:2) 10 9 Avanti 110521 TG(16:0/16:0/18:1) 5 10Avanti 111000 TG(16:0/16:0/16:0) 10 11 Avanti 850468 PC(18:0/18:2) 10 12Avanti 850458C PC(16:0/18:2) 10 13 Avanti 111002 TG(18:2/18:2/18:2) 1014 Avanti 860634P C16 Dihydroceramide 5 (d18:0/16:0) 15 Sigma P8577TG(16:0/18:1/18:2) 1 16 Nu-chek T-160 TG(18:0/18:0/18:0) 1 17 Matreya 1326 So(d16:0) 1 18 Sigma D1782 TG(16:0/18:1/18:1) 5 19 Larodan32-1656-7 DG(16:0/18:2) 5 20 Larodan 34-1603-7 TG(16:0/16:0/18:2) 5 21Larodan 34-1862-7 TG(16:0/18:2/18:2) 5 22 Larodan 34-3003-7TG(18:0/16:0/18:1) 5 23 Larodan 34-1822-7 TG(18:0/18:1/18:1) 5 24Larodan 34-3007-7 TG(18:0/18:1/18:2) 5 25 Larodan 34-1827-7TG(18:1/18:1/18:2) 5 26 Larodan 34-1828-7 TG(18:1/18:1/18:3) 5 27Larodan 34-1866-7 TG(18:1/18:2/18:2) 5 28 Larodan 34-1855-7TG(18:3/18:2/18:2) 5 29 Larodan 10-1840-4 FA(18:4) 5 30 Avanti 110748Sphinganine (d18:0) 5 31 Avanti 110749 Sphinganine (d20:0) 1 32 Avanti110520 TG(18:0/16:0/16:0) 5 33 Larodan 34-1810-7 TG(18:0/16:0/18:0) 1034 Larodan 31-1820-7 MG(18:2p) 10 35 nu-chek D-251 DG(18:2/18:2) 10 36Larodan 38-1802-0 LPC(18:2) 10 37 avanti 791251 LPC(18:3) 10 38 avanti791016 PE(16:0/16:1) 10 39 avanti 792077C 16:1-18:1PE 10 40 avanti792078C 16:0-22:1 PE 10 41 avanti 792079P Sphinganine(d22:0) 10 42Larodan 31-2220 MG(22:2) 10 43 Larodan 32-1658 DG(16:0/18:3) 10 44Larodan 34-1289 TG(18:1/18:1/20:4) 10 45 Larodan 34-1870 DG(18:3/18:2)10 46 Larodan 32-1871 DG(20:5/18:2) 10 47 Larodan 34-1880TG(18:3/18:2/18:3) 10 48 Larodan 34-2230 TG(18:1/22:1/22:1) 10 49Larodan 34-3031 TG(16:0/16:1/18:1) 10 50 Larodan 34-3032TG(16:0/18:1/18:3) 10 51 Larodan 34-3033 TG(16:0/18:1/20:4) 10 52Larodan 34-3034 TG(18:3/18:2/20:5) 10 53 Avanti 792143 Cer(d16:0/16:0)10 54 Avanti 792144 Cer(d20:0/18:0) 10 55 Avanti 792145 Cer(d22:0/18:0)10 56 Avanti 792146 TG(16:0/18:2/18:3) 10 57 Avanti 792147TG(18:1/18:2/18:3) 10 58 Avanti 792150 PEt(16:1/16:1) 10 59 Avanti792151 dMePE(16:1/14:0) 10 60 Avanti 792152 dMePE(16:1/16:1) 10 61Avanti 792153 dMePE(18:1/14:0) 10 62 Avanti 792154 dMePE(16:1/18:1) 1063 Avanti 792156 PC(18:0/18:3(6Z,9Z, 10 12Z)) 64 Avanti 792155PE(15:0/24:1(15Z)) 10 65 Avanti 792157 PC(20:0/14:1(9Z)) 10 66 Avanti792160 TG(18:0/18:2/18:3) 10 67 Avanti 792148 TG(18:1/18:2/20:5) 10 68Avanti 792149 TG(20:5/18:2/18:2) 10 69 Avanti 792158 PC(18:1(11Z)- 1016:1(9Z)) 70 Larodan 32-1830-7 DG(18:3/18:3) 25 71 Larodan 37-1620-7PE(16:0/16:0) 25

TABLE 1-2 Description of lipids 1-32 No. Abbreviation IUPAC nameStructure  1 DG(18:0/18:0/ 0:0) (2S)-1-hydroxy-3- (octadecanoyloxy)propan-2-yl octadecanoate

 2 DG(18:0/16:0/ 0:0) (2S)-2- (hexadecanoyloxy)-3- hydroxypropyloctadecanoate

 3 DG(16:0/16:0/ 0:0) (2S)-1- (hexadecanoyloxy)-3- hydroxypropan-2-ylhexadecanoate

 4 C18 Dihydroceramide (d18:0/18:0) N-[(2S,3R)-1,3- dihydroxyoctadecan-2-yl]octadecanamide

 6 TG(18:1/18:1/ 18:1) 1,3-bis[(9Z)- octadec-9- enoyloxy]propan-2-yl(9Z)-octadec-9-enoate

 8 PE(16:0/18:2) (2- aminoethoxy)[(2R)-3- (hexadecanoyloxy)-2-[(9Z,12Z)-octadeca- 9,12- dienoyloxy]propoxy] phosphinic acid

 9 TG(16:0/16:0/ 18:1) (2R)-2,3- bis(hexadecanoyloxy) propyl(9Z)-octadec- 9-enoate

10 TG(16:0/16:0/ 16:0) 1,3- bis(hexadecanoyloxy) propan-2-ylhexadecanoate

11 PC(18:0/18:2) trimethyl(2-{[(2R)- 2-[(9Z,12Z)-octadeca-9,12-dienoyloxy]-3- (octadecanoyloxy) propyl phosphonato]oxy}ethyl)azanium

12 PC(16:0/18:2) (2-{[(2R)-3- (hexadecanoyloxy)-2- [(9Z,12Z)-octadeca-9,12- dienoyloxy]propyl phosphonato]oxy} ethyl)trimethylazanium

13 TG(18:2/18:2/ 18:2) 1,3-bis[(9Z,12Z)- octadeca-9,12-dienoyloxy]propan-2- yl (9Z,12Z)-octadeca- 9,12-dienoate

14 C18 Dihydroceramide (d18:0/16:0) N-[(2S,3R)-1,3- dihydroxyoctadecan-2-yl]hexadecanamide

15 TG(16:0/18:1/ 18:2) 1-Palmitoyl-2- oleoyl-3-linoleoyl- rac-glycerol

16 TG(18:0/18:0/ 18:0) 1,3- bis(octadecanoyloxy) propan-2-yloctadecanoate

17 So(d16:0) D,L-2- Aminohexadecane- 1,3-diol

18 TG(16:0/18:1/ 18:1) 1,2-Di(cis-9- octadecenoyl)-3- hexadecanoyl-rac-glycerol

19 DG(16:0/18:2) 1-Palmitoyl-3- Linoleoyl-sn-glycerol

20 TG(16:0/16:0/ 18:2) 1,2-Palmitoyl-3- Linoleoyl-sn-glycerol

21 TG(16:0/18:2/ 18:2) 1,2-Linoleoyl-3- Palmitoyl-sn-glycerol

22 TG(18:0/16:0/ 18:1) 1-Stearoyl-2- Palmitoyl-3-Oleoyl- sn-glycerol

23 TG(18:0/18:1/ 18:1) 1,2-olein-3-stearin

24 TG(18:0/18:1/ 18:2) 1-Stearoyl-2- Oleoyl-3-Linoleoyl- sn-glycerol

25 TG(18:1/18:1/ 18:2) 1,2-Oleoyl-3- Linoleoyl-sn-glycerol

26 TG(18:1/18:1/ 18:3) 1,2-Oleoyl-3- Linolenoyl-sn- glycerol

27 TG(18:1/18:2/ 18:2) 1,2-Linoleoyl-3- Oleoyl-sn-glycerol

28 TG(18:3/18:2/ 18:2) 1,2-Linoleoyl-3- Linolenoyl-sn- glycerol

29 FA(18:4) 6c,9c,12c,15c- Octadecatetraenoic Acid

30 Sphinganine (d18:0) (2S,3R)-2- aminooctadecane-1,3- diol

31 Sphinganine (d20:0) (2S,3R)-2-amino- 1,3-eicosanediol

32 TG(18:0/16:0/ 16:0) (2R)-2,3- bis(hexadecanoyloxy) propyloctadecanoate

TABLE 1-2 Description of lipids 33-71 No. Abbreviation IUPAC NameStructure 33 TG(18:0/16:0/ 18:0) 1,3-Stearin-2- Palmitin1,3-Octadecanoyl- 2-Palmitoyl- glycerol

34 MG(18:2p) (9Z,12Z)- Octadeca-9,12- dienoic acid, monoester withglycerol 9,12- Octadecadienoic acid (9Z,12Z)-, monoester with1,2,3-propanetriol 9,12- Octadecadienoic acid, (Z,Z)-, monoester with1,2,3-propanetriol

35 DG(18:2/ 18:2) (2S)-1-hydroxy-3- [(9Z,12Z)-octadeca- 9,12-dienoyloxy]propan- 2-yl (9Z,12Z)- octadeca-9,12- dienoate ,

36 LPC(18:2) 1-Linoleoyl-2- Hydroxy-sn- Glycero-3- Phosphatidylcholine

37 LPC(18:3) (2-{[(2R)-2- hydroxy-3- [(9Z,12Z,15Z)- octadeca-9,12,15-trienoyloxy]propyl- phosphonato]oxy} ethyl)trimethylazanium

38 PE(16:0/ 16:1) (2- aminoethoxy)[(2R)- 2-[(9Z)-hexadec-9- enoyloxy]-3-(hexadecanoyloxy) propoxy]phosphinic acid

39 16:1- 18:1PE (2- aminoethoxy)[(2R)- 3-[(9Z)-hexadec-9-enoyloxy]-2-[(11Z)- octadec-11- enoyloxy]propoxy] phosphinic acid

40 16:0-22:1 PE (2- aminoethoxy)[(2R)- 2-[(13Z)-docos-13- enoyloxy]-3-(hexadecanoyloxy) propoxy]phosphinic acid

41 Sphinganine (d22:0)

42 MG(22:2) Monodocosadienoin

43 DG(16:0/ 18:3) 1-Palmitin-3- Linolenin

44 TG(18:1/18:1/ 20:4) 1,2-Olein-3- Arachidonin(5Z,8Z, 11Z,14Z)

45 DG(18:3/ 18:2) 1-Linolein-3- Linolenin

46 DG(20:5/ 18:2) 1-EPA-3-Linolein

47 TG(18:3/18:2/ 18:3) 1,3-Linolenin-2- Linolein

48 TG(18:1/22:1/ 22:1) 1,2-Eucin(13Z)-3- Olein

49 TG(16:0/16:1/ 18:1) 1-Palmitin-2- Palmitolein-3-Olein

50 TG(16:0/18:1/ 18:3) 1-Palmitin-2- Olein-3-Linolenin

51 TG(16:0/18:1/ 20:4) 1-Palmitin-2- Olein-3- Arachidonin(5Z,8Z,11Z,14Z)

52 TG(18:3/18:2/ 20:5) 1-Linolenin-2- Linolein-3-EPA

53 Cer(d16:0/ 16:0) C16 dihydroceramide (d16:0/16:0)

54 Cer(d20:0/ 18:0) C18 dihydroceramide (d20:0/18:0)

55 Cer(d22:0/ 18:0) C18 dihydroceramide (d22:0/18:0)

56 TG(16:0/18:2/ 18:3) Triglyceride(16:0/ 18:2/18:3)

57 TG(18:1/18:2/ 18:3) Triglyceride(18:1/ 18:2/18:3)

58 PEt(16:1/ 16:1) 1,2- dipalmitoleoyl-sn- glycero-3- phosphoethanol

59 dMePE(16:1/ 14:0) 1-palmitoleoyl-2- myristoyl-sn- glycero-3-phosphoethanolamine- N,N-dimethyl

60 dMePE(16:1/ 16:1) 1,2- dipalmitoleoyl-sn- glycero-3-phosphoethanolamine- N,N-dimethyl

61 dMePE(18:1/ 14:0) 1-oleoyl-2- myristoyl-sn- glycero-3-phosphoethanolamine- N,N-dimethyl

62 dMePE(16:1/ 18:1) 1-palmitoleoyl-2- oleoyl-sn-glycero-3-phosphoethanolamine- N,N-dimethyl

63 PC(18:0/18:3 (6Z,9Z,12Z)) 1-stearoyl-2- linolenoyl(gamma)-sn-glycero-3- phosphocholine

64 PE(15:0/24:1 (15Z)) 1-pentadecanoyl- 2-nervonoyl-sn- glycero-3-phosphoethanolamine

65 PC(20:0/14:1 (9Z)) 1-eicosanoyl-2- myristoleoyl-sn- glycero-3-phosphocholine

66 TG(18:0/18:2/ 18:3) Triglyceride(18:0/ 18:2/18:3)

67 TG(18:1/18:2/ 20:5) Triglyceride(18:1/ 18:2/20:5)

68 TG(20:5/18:2/ 18:2) Triglyceride(20:5/ 18:2/18:2)

69 PC(18:1(11Z)- 16:1(9Z)) 1-vaccenoyl-2- palmitoleoyl-sn- glycero-3-phosphocholine

70 DG(18:3/ 18:3) Dilinolenin

71 PE(16:0/16:0) 1,2-Dipalmitoyl- sn-Glycero-3- Phosphatidylethanolamine

Definition of Terms

The term as used herein may have a single dash “-” (or horizontal line)or a double dash “=” in front of and/or behind it to indicate the bondlevel of the bond between the mentioned substituent and its parentmoiety; a single dash “-” (or horizontal line) refers to a single bond,and a double dash refers to a double bond; in the absence of single ordouble dash, it is understood that a single bond is formed between thesubstituent and its parent moiety; in addition, the substituent is to beconstrued “from left to right” unless the dash indicatesotherwise; forexample, a C1-C6 alkoxycarbonyloxy group and an —OC(O)OC1-C6 alkyl grouprefer to the same functional group.

The term “alkyl” as used herein refers to a straight or branchedsaturated hydrocarbon chain. As described herein, an alkyl group has 1to 20 carbon atoms (i.e., C1-20 alkyl), 1 to 8 carbon atoms (i.e., C1-8alkyl), 1 to 6 carbon atoms (i.e., C1-6 alkyl), or 1 to 4 carbon atoms(i.e., C1-4 alkyl). In one embodiment, the alkyl group is a C10-20 alkylgroup. In one embodiment, the alkyl group is a C15-20 alkyl group. Inone embodiment, the alkyl group is a C15-18 alkyl group, i.e., a C15,C16, C17, C18 alkyl group.

The term “alkenyl” as used herein refers to an aliphatic groupcontaining at least one carbon-carbon double bond and having 2 to 20carbon atoms (i.e., C2-20 alkenyl), 2 to 8 carbon atoms (i.e., C2-8alkenyl), 2 to 6 carbon atoms (i.e., C2-6 alkenyl) or 2 to 4 carbonatoms (i.e., C2-4 alkenyl). In one embodiment, the alkenyl group is aC10-20 alkenyl group. In one embodiment, the alkenyl group is a C15-20alkenyl group. In one embodiment, the alkenyl group is a C15-18 alkenylgroup, i.e. a C15, C16, C17, C18 alkenyl group.

The term “heteroalkyl” and “heteroalkenyl” as used herein refer to alkyland alkenyl as defined above, respectively, wherein one or more carbonatoms are each independently substituted by the same or differentheteroatom groups. For example, 1, 2 or 3 carbon atoms may beindependently substituted by the same or different heteroatom groups.Heteroatom groups include, but are not limited to, —NR1-, —O—, —S—,—S(O)—, —S(O)2-, and the like, wherein R1 is H, alkyl. Examples ofheteroalkyl groups include —OCH3, —CH2OCH3, —SCH3, —CH2SCH3, —NR1CH3 and—CH2NR1CH3, wherein R1 is hydrogen, alkyl.

The term reverse evaporation method as described herein refers to addingan aqueous solution of nucleic acid to an organic solvent solution oflipid, ultrasonicating, evaporating to remove the organic solvent, andthen hydrating to obtain a lipid nucleic acid mixture.

The term “boiling method” (also refers to “heating method”) as describedherein refers to adding an organic solvent solution of lipid to anaqueous solution of nucleic acid and boiling at about 100° C. for 30minutes to obtain a lipid nucleic acid mixture. The method is notlimited to heating by boiling, and other means of heating or raisingtemperature known in the art can also be used.

Reverse evaporation method and boiling method are carried out undercontrolled temperature and mixing conditions. Suitable processing times,and temperatures can be readily determined by a person skilled in theart. For example, the temperature of reverse evaporation method isranged preferably from about 25° C. to about 70° C., more preferablyfrom about 30° C. to about 65° C., and more preferably from about 40° C.to about 60° C., especially about 55° C. The temperature of boilingmethod is ranged preferably from about 25° C. to about 100° C., morepreferably from about 50° C. to about 100° C., and more preferably fromabout 95° C. to about 100° C., especially preferably from about 80° C.to 100° C.

The nucleic acid as described herein comprises DNA and RNA, preferablysmall RNA, for example, the small RNA having a length of 14-32 bp, 16-28bp, 18-24 bp, and particularly, a length of 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 bp.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1: Effect of 12 lipids on nucleic acid (HJT-sRNA-m7) absorption andentry into cells (human gastric cancer cell line NCI-N87) (reverseevaporation method).

FIG. 2: 27 single lipidspromote nucleic acid entry into MRC-5 cell line(reverse evaporation method).

FIG. 3: 23 single lipids promote nucleic acid entry into MRC-5 cell line(boiling method).

FIG. 4: 23 single lipids promote nucleic acid entry into A549 cell line(boiling method).

FIG. 5: Lipid combination can promote nucleic acid entry into MRC-5 cellline (reverse evaporation method).

FIG. 6: Lipid combination can promote nucleic acid entry into A549 cellline (reverse evaporation method).

FIG. 7: Lipid combination can promote nucleic acid entry into MRC-5 cellline (boiling method).

FIG. 8: Lipid combination can promote nucleic acid entry into A549 cellline (boiling method).

FIG. 9: Different types of lipid combinations promote nucleic acid entryinto Caco-2 cell line (reverse evaporation method).

FIG. 10: Different types of lipid combinations promote nucleic acidentry into Caco-2 cell line (boiling method).

FIG. 11A-C: Single lipids (No. 11 and No. 12) promote nucleic acidshaving different sequences entry into different cells.

FIG. 12: Fluorescence in situ hybridization experiment indicates thatthe nucleic acids enter into the cytoplasm with the aid of single lipid.

FIG. 13: Single lipids (No. 11 and No. 12) promote nucleic acid entryinto cells, targeting the gene 3′UTR region.

FIG. 14: Single lipids (No. 11 and No. 12) promote nucleic acid entryinto blood and lung by digestive tract.

FIG. 15: Lipid combinations prepared by reverse evaporation method andboiling method facilitate nucleic acid entry into blood and lung bydigestive tract.

FIG. 16: Different types of lipid combinations deliver single-strandednucleic acid into MRC-5.

FIG. 17A-B: Lipid combinations deliver single-stranded nucleic acid intoMRC-5 or Caco-2 cells.

FIG. 18: Lipid combinations deliver single-stranded nucleic acid intocells.

FIG. 19: Lipid combinations deliver single-stranded nucleic acid intocells.

FIG. 20: Lipid combinations deliver single-stranded nucleic acid intocells.

FIG. 21: Lipid combinations deliver single-stranded nucleic acid intoA549 cell.

FIG. 22: Lipid combinations deliver single-stranded nucleic acid intoA549 cell.

FIG. 23: Lipid combinations deliver single-stranded nucleic acid intoA549 cell.

FIG. 24: Lipid combinations deliver single-stranded nucleic acid intoA549 cell.

FIG. 25: Lipid combinations deliver single-stranded nucleic acid intoA549 cell.

FIG. 26: Lipid combinations deliver single-stranded nucleic acid intoA549 cell.

FIG. 27: Lipid combinations deliver single-stranded nucleic acid intoA549 cell.

FIG. 28: Lipid combinations deliver single-stranded nucleic acid intoA549 cell.

FIG. 29: Lipid combinations deliver single-stranded nucleic acid intoA549 cell.

FIG. 30: Lipid combinations deliver single-stranded nucleic acid intoA549 cell.

FIG. 31: Lipid combinations deliver single-stranded nucleic acid intoA549 cell.

FIG. 32: Lipid combinations deliver single-stranded nucleic acid intoA549 cell.

FIG. 33: Lipid combinations deliver double-stranded nucleic acid intoMRC-5 cell.

FIG. 34: Lipid combinations deliver double-stranded nucleic acid intoMRC-5 cell.

FIG. 35: Lipid combinations deliver double-stranded nucleic acid intoA549 cell.

FIG. 36: Lipid combinations deliver double-stranded nucleic acid intoA549 cell.

FIG. 37: Lipid combinations deliver double-stranded nucleic acid intoA549 cell.

FIG. 38: Lipid combinations deliver double-stranded nucleic acid intoA549 cell.

FIG. 39: Lipid combinations deliver double-stranded nucleic acid intoA549 cell.

FIG. 40: Lipid combinations deliver double-stranded nucleic acid intoA549 cell.

FIG. 41: Lipid combinations deliver double-stranded nucleic acid intoA549 cell.

FIG. 42: Lipid combinations deliver double-stranded nucleic acid intoA549 cell.

FIG. 43: Lipid combinations deliver double-stranded nucleic acid intoMRC-5 cell.

FIG. 44: Lipid combinations deliver double-stranded nucleic acid intoMRC-5 cell.

FIG. 45: Lipid combinations deliver double-stranded nucleic acid intoMRC-5 cell.

FIG. 46: Lipid combinations deliver double-stranded nucleic acid intoMRC-5 cell.

FIG. 47: Lipid combinations deliver double-stranded nucleic acid intoMRC-5 cell.

FIG. 48: Lipid combinations deliver double-stranded nucleic acid intoMRC-5 cell.

FIG. 49: Lipid combinations deliver double-stranded nucleic acid intoMRC-5 cell.

FIG. 50: Lipid combinations promote nucleic acid entry into lung viadigestive tract.

FIG. 51: No. 8(PE):No. 12(PC) (v:v=1:2) mediates anti-fibroticHJT-sRNA-m7 entry into MRC-5 cell.

FIG. 52: No. 8(PE):No. 12(PC) (v:v=1:2) mediates siRNA entry into A549cell.

FIG. 53: No. 8(PE):No. 12(PC) (v:v=1:2) mediates siRNA entry into A549cell.

FIG. 54: No. 8(PE):No. 12(PC) (v:v=1:2) mediates siRNA entry into THP-1cell.

FIG. 55: No. 8(PE):No. 12(PC):No. 2(DG) (v:v:v=2:4:3) mediatesanti-fibrotic HJT-sRNA-m7 entry into MRC-5 cell.

FIG. 56: No. 8(PE):No. 12(PC):No. 2(DG) (v:v:v=2:4:3) lipid mixturemediates XRN2 siRNA entry into A549 cell to inhibit gene expression.

FIG. 57: No. 8(PE):No. 12(PC):No. 4(Cer) (v:v:v=1:2:1) lipid mixturemediates anti-fibrotic HJT-sRNA-m7 entry into MRC-5 cell (boilingmethod).

FIG. 58: No. 8(PE):No. 12(PC):No. 4(Cer) (v:v:v=1:2:1) lipid mixturemediates NFκB siRNA entry into THP-1 cell to inhibit gene expression(boiling method).

FIG. 59: No. 8(PE):No. 12(PC):No. PC(11) (v:v:v=1:2:1) lipid mixturemediates XRN2 siRNA entry into A549 cell to inhibit gene expression.

FIG. 60: No. 8(PE):No. 12(PC):No. LPC(37) (v:v:v=1:2:1) lipid mixturemediates XRN2 siRNA entry into A549 cell to inhibit gene expression.

FIG. 61: No. 8(PE):No. 12(PC):No. MG(34) (v:v:v=2:3:1) lipid mixturemediates CPSF4 siRNA entry into A549 cell to inhibit gene expression.

FIG. 62: No. 38(PE):No. 37(LPC):No. 32(TG) (v:v:v=32:8:5) lipid mixturemediates anti-fibrotic HJT-sRNA-m7 entry into MRC-5 cell (boilingmethod).

FIG. 63: No. 38(PE):No. 37(LPC):No. 32(TG) (v:v:v=32:8:5) lipid mixturemediates XRN2 siRNA entry into A549 to inhibit gene expression.

FIG. 64: No. 1(DG):No. 8(PE):No. 12(PC):No. 4(Cer):No. 31(So):No.29(FA):No. 16(TG) (v:v:v:v:v:v:v=2:1:2:2:3:1:3) mediates anti-fibroticHJT-sRNA-m7 entry into MRC-5 cell (boiling method).

FIG. 65: No. 1(DG):No. 8(PE):No. 12(PC):No. 4(Cer):No. 31(So):No.29(FA):No. 16(TG) (v:v:v:v:v:v:v=2:1:2:2:3:1:3) lipid mixture mediatesXRN2 siRNA entry into A549 to inhibit gene expression (boiling method).

FIG. 66: No. 8(PE):No. 12(PC):No. 31(So):No. 29(FA):No. 4(Cer)(v:v:v:v:v=2:4:2:2:2:5) mediates anti-fibrotic HJT small RNA HJT-sRNA-3,HJT-sRNA-a2, HJT-sRNA-h3, HJT-sRNA-m7 entry into MRC-5 cell (boilingmethod).

FIG. 67: No. 8(PE):No. 12(PC):No. 31(So):No. 29(FA):No. 4(Cer)(v:v:v:v:v=2:4:2:2:5) lipid mixture can effectively deliver nucleic acidinto cell.

FIG. 68: No. 38(PE):No. 37(LPC) (v:v=4:1) mediates anti-fibrotic HJTsmall RNA HJT-sRNA-3, HJT-sRNA-a2, HJT-sRNA-h3, HJT-sRNA-m7 entry intoMRC-5 cell (boiling method).

FIG. 69: No. 38(PE):No. 37(LPC) (v:v=4:1) lipid mixture mediates XRN2siRNA entry into A549 cell to inhibit gene expression (boiling method).

FIG. 70: No. 38(PE):No. 12(PC):No. 2(DG) (v:v:v=4:1:3) lipid mixturemediates XRN2 siRNA entry into A549 cell to inhibit gene expression.

FIG. 71: No. 38(PE):No. 37(LPC):No. 12(PC) (v:v:v=4:1:1) lipid mixturemediates XRN2 siRNA entry into A549 cell to inhibit gene expression(reverse evaporation method).

FIG. 72: No. 4(Cer):No. 12(PC):No. 38(PE):No. 37(LPC) (v:v:v:v=5:2:8:3)lipid mixture mediates anti-fibrotic small RNA HJT-sRNA-3, HJT-sRNA-a2,HJT-sRNA-h3, and HJT-sRNA-m7 entry into MRC-5 cell (boiling method).

FIG. 73: No. 4(Cer):No. 12(PC):No. 38(PE):No. 37(LPC) (v:v:v:v=5:2:8:3)lipid mixture mediates XRN2 siRNA entry into A549 cell to inhibit geneexpression (boiling method).

FIG. 74: No. 38(PE):No. 2(DG):No. 31(So) (v:v:v=4:2:3) lipid mixturemediates anti-fibrotic small RNA HJT-sRNA-3, HJT-sRNA-a2, HJT-sRNA-h3,HJT-sRNA-m7 entry into MRC-5 cell (boiling method).

FIG. 75: No. 38(PE):No. 2(DG):No. 31(So) (v:v:v=4:2:3) lipid mixturemediates XRN2 siRNA entry into A549 cell to inhibit gene expression(boiling method).

FIG. 76: Lipid No. 41 delivers double-stranded RNA into A549 cell bydifferent preparation methods (boiling or reverse evaporation method).

FIG. 77: Lipid No. 41 delivers double-stranded RNA into MRC-5 cell bydifferent preparation methods (boiling or reverse evaporation method).

FIG. 78: Lipid No. 41 delivers single-stranded RNA into A549 and MRC-5cells by boiling method.

FIG. 79: Digital PCR (ddPCR) technology determines the efficiency ofnucleic acid delivery by lipid.

FIG. 80: Flow cytometry technology determined the efficiency of nucleicacid delivery by lipid.

FIG. 81: Confocal fluorescence microscopy observes the localization ofnucleic acid delivered by lipid in cell.

FIG. 82: Western Blotting assay determined the efficiency of nucleicacid delivery by lipid.

FIG. 83: Single lipid No. 41 mediates anti-fibrotic HJT-sRNA-m7 entryinto MRC-5 cell (boiling method).

FIG. 84: Effects of lipid combination 1 (No. 8+No. 41=6:1) and lipidcombination 2 (No. 38+No. 41=6:1) in nucleic acid delivery.

FIG. 85: Effects of lipid combination 3 (No. 39+No. 41=6:1) and lipidcombination 4 (No. 40+No. 41=6:1) in nucleic acid delivery.

FIG. 86: Effects of lipid combination 5 (38+12+41+29=1:2:1:1) in nucleicacid delivery.

FIG. 87: Effects of lipid combination 6 (40(PE)+12(PC)+41(So)=2:4:3) innucleic acid delivery.

FIG. 88: Effects of lipid combination 7 (12(PC)+41(So)=1:6) and lipidcombination 8 (12(PC)+41(So)=1:1) in nucleic acid delivery.

FIG. 89: Effects of lipid combination 9 (12(PC)+41(So)=6:1) and lipidcombination 10 (40(PE)+12(PC)+41(So)=2:2:2) in nucleic acid delivery.

FIG. 90: Effects of lipid combination 11 (4(Cer)+12(PC)+41(So)=1:1:1) innucleic acid delivery.

FIG. 91: Lipid 38 delivers double-stranded RNA into A549 and MRC-5 cellsby boiling method.

FIG. 92: Lipid 38 delivers single-stranded RNA into A549 cells and MRC-5cells by boiling method.

FIG. 93: Digital PCR (ddPCR) technology determined the efficiency ofnucleic acid delivery by lipid.

FIG. 94: Flow cytometry technology determined the efficiency of nucleicacid delivery by lipid.

FIG. 95: Confocal fluorescence microscopy observes the location ofnucleic acid delivered by lipid in cell.

FIG. 96: Lipid 64 delivers double-stranded RNA into A549 cell bydifferent preparation methods (boiling or reverse evaporation method).

FIG. 97: The efficiency of nucleic acid delivery by lipid as determinedby flow cytometry technology.

FIG. 98: The localization of nucleic acid delivered by lipid in cell asobserved by confocal fluorescence microscopy.

FIG. 99: The efficiency of nucleic acid delivery by lipid as determinedby Digital PCR (ddPCR).

FIG. 100: The location of nucleic acid delivered by lipid in cell asobserved by confocal fluorescence microscopy.

FIG. 101: The efficiency of nucleic acid delivery by lipid as determinedby Western Blotting assay.

FIG. 102: Single phosphatidylethanolamine lipid 40 mediatesanti-fibrotic double-stranded RNA HJT-sRNA-m7 entry into MRC-5 cell todown-regulate fibronectin protein expression level.

FIG. 103: Lipid 38 prepared by boiling method delivers single-strandedRNA into A549 and MRC-5 cells.

FIG. 104: Lipid 39 prepared by different methods (boiling or reverseevaporation method) delivers double-stranded RNA into A549 cell.

FIG. 105: The efficiency of nucleic acid delivery by lipid determined byDigital PCR (ddPCR).

FIG. 106: Lipid 60 prepared by different methods (boiling or reverseevaporation method) delivers double-stranded RNA into A549 cell.

FIG. 107: Lipid 62 prepared by different methods (boiling or reverseevaporation method) delivers double-stranded RNA into A549 cell.

FIG. 108: Lipid No. 41 promotes small RNA entry into blood and protectsit from degradation in the blood.

FIG. 109: Lipid No. 41 promotes small RNA entry into stomach cell andprotects it from degradation in the stomach.

FIG. 110: Lipid No. 41 promotes small RNA entry into small intestinecell and protects it from degradation in the small intestine.

FIG. 111: Lipid No. 41 promotes small RNA entry into liver and protectsit from degradation in the liver.

FIG. 112: Single PE (No. 38) effectively delivers single-stranded sRNAnucleic acid into mouse blood by oral administration.

FIG. 113: Single PE (No. 40) effectively delivers single-stranded sRNAnucleic acid into mouse blood by oral administration.

FIG. 114: Single PE (No. 64) effectively delivers single-stranded sRNAnucleic acid into mouse blood by oral administration.

FIG. 115: Single PE (No. 71) effectively delivers single-stranded sRNAnucleic acid into mouse blood by oral administration.

FIG. 116: Lipids effectively deliver single-stranded nucleic acid intoMRC-5 cell at different temperature gradients.

DETAILED DESCRIPTION OF THE INVENTION

The following is a further description of the present application, butis not intended to limit the invention in any way, and any changes madebased on the teachings of the present application fall within the scopeof protection of the present application.

In the present application, lipid-soluble components were extracted fromtraditional Chinese medicines (including Rhodiola crenulata, Taraxacummongolicum, Andrographis paniculata and Lonicera japonica) by theBligh&Dyer method, and the lipid components were identified byHPLC-MS/MS (a total of 138 lipid components were identified, 125 inpositive mode, 13 in negative mode). 71 of them (see Table 1-1 to Table1-3) were used for the preparation of the lipid nucleic acid mixtures,and observed for whether they could promote cellular absorption andentry of exogenous nucleic acids. It should be noted that the lipidsused in the present application were commercially purchased orcommercially synthesized, and were not directly extracted fromtraditional Chinese medicines. The inventor has surprisingly found thatvarious lipids can form lipid nucleic acid mixtures that effectivelypromote cellular absorption and entry of nucleic acid (see FIGS. 1-116),having the potential of increasing the efficiency of the nucleic aciddrug delivery in clinical settings. Further studies have shown that thelipid nucleic acid mixture of the present application promotes theefficiency of nucleic acid absorption and entry in different cell lines,but differences were observed in different cell lines (see FIGS. 1-10),which opens up the possibility of targeted drug delivery. Moreover,nucleic acid delivery by such lipid nucleic acid mixture is not sequencespecific, capable of delivering nucleic acid fragments having differentsequences and a size corresponding to that of small RNA (e.g. about 20bp) (see FIG. 11). In addition, fluorescence in situ hybridization(FISH) confirmed that the lipid nucleic acid mixture formed by lipidsderived from the decoction can effectively promote the entry ofexogenous nucleic acids into cytoplasm (see FIG. 12). The inventor hasunexpectedly discovered that lipid nucleic acid mixtures prepared byboiling or reverse evaporation method can facilitate entry of nucleicacids, such as sRNA, into blood circulation and target tissue vianon-invasive routes (e.g. via digestive tract, respiratory tract andtopical administration). (See FIGS. 14-15). The inventor has alsosurprisingly discovered that lipids of the present application arecapable of promoting entry of nucleic acids, such as sRNA, into cellsand modulating (e.g., inhibiting) the expression of their targetsequences, while not exhibiting such regulatory effects on non-targetsequences, suggesting a target-specific regulation, which can be used asa means for the delivery of nucleic acid drug (see FIG. 13).

Based on the above unexpected discoveries, the inventors have arrived atthe present application.

In one aspect, the present application provides compounds extracted fromtraditional Chinese medicines for facilitating nucleic acid delivery,wherein the said compounds are selected from the group consisting oflysolecithin, ceramide, diglyceride, phosphatidylethanolamine,phosphatidylcholine, triglyceride, monogalactosyl diglycerides,sphingosine, phosphatidyl ethanol, monoacylglycerol, fatty acid,platelet activating factor, or dimethyl phosphatidyl ethanolamine,preferably selected from the lipids shown in Table 1. In one embodiment,the lipid is non-natural, e.g. synthetic, or manufactured fromfermentation.

In one embodiment, the lipid is used to deliver a nucleic acid into atarget cell. In another embodiment, the lipid is used to deliver anucleic acid into a subject in need thereof and into its bloodcirculation and/or a target site/cell.

In a preferred embodiment, the lipid is selected fromphosphatidylcholine, e.g.,1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine (PC(18:0/18:2), i.e.,lipid No. 11 in Table 1), and1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (PC(16:0/18:2), i.e.,lipid No. 12 in Table 1). These two phosphocholine (PC) are capable ofefficiently encapsulating nucleic acids or promoting entry of nucleicacids into cells. In one embodiment, the lipid may be lipid No. 41 inTable 1, i.e. sphinganine(d22:0), which is capable of efficientlyencapsulating nucleic acids or promoting entry of nucleic acids intocells.

In another aspect, the present application provides pharmaceuticalcompositions comprising the above lipids and nucleic acids. Preferablythe nucleic acid is small RNA.

In one embodiment, the pharmaceutical composition of the presentapplication can be prepared for administration via non-invasive routes(e.g., topical administration) and/or injection, e.g., administrationvia digestive tract, respiratory tract, and/or injection, e.g., oraladministration, inhalation and/or injection. In some cases, invasiveroutes are preferred (e.g., injection, including intramuscular,subcutaneous, intravenous, intraarterial, intraperitoneal, and injectioninto a target tissue; in other cases, non-invasive routes are preferred.

In another embodiment, in the pharmaceutical composition of the presentapplication, at least part of or all of the lipids and nucleic acids canbe prepared into the form of lipid nucleic acid mixture. Various methodsfor the manufacture of lipid nucleic acid mixtures have been widelydisclosed, and the suitable method for the manufacture of lipid nucleicacid mixture can be selected according to actual needs.

In a third aspect, the present application provides kits comprising thelipids and nucleic acids described herein, wherein the lipids and thenucleic acids are each independently provided in a first container and asecond container. The first container and the second container may bethe same or different. In some embodiments, at least part of or all ofthe lipids and the nucleic acids are prepared into lipid nucleic acidmixtures immediately prior to use.

In a fourth aspect, the present application provides methods ofdelivering a nucleic acid into a target tissue/cell, wherein the nucleicacid is provided in a form of the pharmaceutical composition or the kitas described herein.

In a fifth aspect, the present application provides methods ofdelivering a nucleic acid into a subject in vivo in need thereof,wherein the nucleic acid is provided in a form of the pharmaceuticalcomposition or the kit as described herein, e.g., delivering the nucleicacid into blood circulation or a target tissue/cell of the subject invivo, e.g., wherein the lipid and the nucleic acid are administrated bynon-invasive routes (e.g., topical administration) and/or injection,e.g., by digestive tract, respiratory tract and/or injection, e.g., byoral administration, inhalation and/or injection.

In a sixth aspect, the present application provides methods ofpreventing and/or treating a disease/disorder that can be preventedand/or treated with a nucleic acid, the methods comprising providing thepharmaceutical composition or the kit described herein to a subject inneed thereof, e.g., wherein the lipid and the nucleic acid areadministered by non-invasive routes (e.g., topical administration)and/or by injection, e.g., by digestive tract, respiratory tract and/orinjection, e.g., by oral administration, inhalation and/or injection.Surprisingly, the non-invasive routes of administration (e.g., bydigestive tract, respiratory tract, including oral administration,gavage, inhalation and the like) can significantly promote the entry andefficacy of nucleic acids.

In a seventh aspect, the present application provides methods for themanufacture of the pharmaceutical composition or the kit, and use of thepharmaceutical composition and/or the kit in the methods described inthe above aspects. Besides, also provided arelipids, pharmaceuticalcompositions and/or kits for use in the various methods described above.

In various embodiments of the present application, the nucleic acid maybe a small RNA, for example, the small RNA may have a length of 14-32bp, 16-28 bp, 18-24 bp, in particular, a length of 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 bp. In addition,the small RNA may be single-stranded, e.g., having a stem-loopstructure, or double-stranded. For example, the nucleic acid may beHJT-sRNA-m7 having the following sequence: ugagguagua gguugugugguuguaagc (SEQ ID NO: 20).

In one embodiment, the pharmaceutical compositions or the kits or thecompounds of the present application can be used for treating a disease,such as cancer, e.g., gastric cancer, lung cancer, and the like.

In one embodiment, the pharmaceutical compositions or the kits or thecompounds of the present application can be used for treating in vitroor in vivo, e.g., to inhibit the growth of NCI-N87 cell (gastric cancercell), MRC-5 cell (lung fibroblast) and A549 cell (lung cancer cell).

In various embodiments of the present application, the lipid nucleicacid mixture can be obtained by a variety of methods, e.g., reverseevaporation method or boiling method. In the reverse evaporation method,an aqueous solution of nucleic acid is added to an organic solventsolution of lipid, ultrasonicated, evaporated to remove the organicsolvent, and then hydrated to obtain a lipid nucleic acid mixture. Theboiling method described in the present application refers to adding anorganic solvent solution of lipid to an aqueous solution of nucleic acidand boiling at about 100° C. for 30 minutes to obtain a lipid nucleicacid mixture. The reverse evaporation method and the boiling method arecarried out under controlled temperature and mixing conditions. Suitableprocessing times and temperatures can be readily determined by a personskilled in the art. For example, the temperature of reverse evaporationmethod can range preferably from about 25° C. to about 70° C., morepreferably from about 30° C. to about 65° C., more preferably from about40° C. to about 60° C., especially preferably about 55° C. Thetemperature of the boiling method (also referred to as heating) canrange preferably from about 25° C. to about 100° C., more preferablyfrom about 50° C. to about 100° C., more preferably from about 95° C. toabout 100° C., especially preferably about 100° C.

Exemplary embodiments of the present application include, but are notlimited to, the following:

Embodiment 1. Use of a compound derived naturally (including atraditional Chinese medicine extract) or synthetically having thefollowing formula for the manufacture of a nucleic acid deliveryreagent, wherein the extract has the structure of the following formulaor comprises a compound having the structure of the following formula:

wherein L1, L2, or L3 is absent, or L1, L2, and L3 are eachindependently selected from the group consisting of —C(O)O—CH2-,—CH(OH)—, —C(O)—NH—CH2-, —CH2-OC(O)—, —CH2-NH—C(O)—, —C(O)O—, —C(O)NH—,—OC(O)—, —NH—C(O)—, —CH2-,

with the proviso that at most two of L1, L2 and L3 are absent;

with respect to the divalent groups L1 and L2, the dash “-” on the leftside is linked to the groups A and B, respectively, and the dash “-” onthe right side is linked to the central carbon atom;

with respect to the divalent group L3, the dash “-” on the left side islinked to the central carbon atom, and the dash “-” on the right side islinked to the group Q;

A, B and Q are each independently selected from the group consisting ofH, —OH, C1-20 alkyl, C1-20 alkenyl, C1-20 heteroalkyl, C1-20heteroalkenyl, —NH2, and —NR3+, R is H or C1-6 alkyl; and n is aninteger 0, 1, 2, 3 or 4;

wherein preferably, the nucleic acid is a small nucleic acid, preferablyis single stranded or double stranded, preferably the length of thesmall nucleic acid is 14-32 bp, 16-28 bp or 18-24 bp;

preferably, the traditional Chinese medicine is selected from the groupconsisting of decoction pieces of Rhodiola crenulata, Taraxacummongolicum, Andrographis paniculata and Lonicera japonica, preferablythe extract is obtained by extracting a lipid-soluble component by theBligh & Dyer method, and more preferably by soaking the Chinese medicinedecoction pieces in water, and then sequentially performing intenseheating and slow heating, and the heated Chinese medicine soup isconcentrated, and then is sequentially added with chloroform-methanol,chloroform and water for stirring, and the chloroform layer is obtained;

preferably, the reagent is an oral reagent; preferably, the nucleic acidis used for treating a disease, such as cancer, for example gastriccancer or lung cancer.

Embodiment 2. The use of embodiment 1, wherein in said structure

L1 is absent, or L1 is selected from —C(O)O—CH2- and —CH(OH)—,

L2 is absent, or L2 is selected from —C(O)O— and —C(O)NH—,

L3 is absent, or L3 is selected from the group consisting of —C(O)O—,—CH2-OC(O)—, —CH2- and

A is selected from the group consisting of H, C1-20 alkyl and C1-20alkenyl;

B is selected from the group consisting of H, —NH2, C1-20 alkyl andC1-20 alkenyl;

Q is selected from the group consisting of H, —OH, C1-20 alkyl and C1-20alkenyl, and —NR3+, wherein R is H or C1-6 alkyl.

Embodiment 3. The use of embodiment 1 or 2, wherein the said compoundhas a structure of the following formula:

Embodiment 4. The use of any one of preceding embodiments, wherein inthe structure

A is selected from the group consisting of H, C10-20 alkyl and C10-20alkenyl;

B is selected from the group consisting of H, —NH2, C10-20 alkyl andC10-20 alkenyl;

Q is selected from the group consisting of H, —OH, C10-20 alkyl andC10-20 alkenyl, and —NR3+, wherein R is H or C1-4 alkyl.

Embodiment 5. The use of embodiment 4, wherein in said structure:

A is selected from the group consisting of H, a straight-chain C15-18alkyl group and a straight-chain C15-18 alkenyl group;

B is selected from the group consisting of H, —NH2, a straight-chainC15-18 alkyl group and a straight-chain C15-18 alkenyl group;

Q is selected from the group consisting of H, —OH, a straight-chainC15-18 alkyl group and a straight-chain C15-18 alkenyl group, and —NR3+wherein R is H or a C1-4 alkyl group; and the alkenyl group in the A, B,Q has 1-5 double bonds.

Embodiment 6. The use of embodiment 5, wherein in the A, B, Q of thesaid structure, the alkenyl group has 1-4 double bonds and is in a Zconfiguration.

Embodiment 7. The use of embodiment 6, wherein the alkenyl group in theA, B,

Q has 1-3 double bonds and is in a Z configuration.

Embodiment 8. The use of any one of the preceding embodiments, whereinsaid extract is selected from the following formulas or comprises acompound selected from the following formulas:

wherein

A is selected from a straight-chain C15-18 alkyl group and astraight-chain C15-18 alkenyl group;

B is selected from a straight-chain C15-18 alkyl group and astraight-chain C15-18 alkenyl group;

Q is selected from the group consisting of H, —OH, a straight-chainC15-18 alkyl group and a straight-chain C15-18 alkenyl group, and —NR3+wherein R is H or methyl;

and

L3 is —C(O)O—.

Embodiment 9. The use of any one of the preceding embodiments, whereinthe extract is or comprises lysolecithin, ceramide, diglyceride,phosphatidylethanolamine, phosphatidylcholine, triglyceride,monogalactosyl diglyceride, (neuro) sphingosine, phosphatidyl ethanol,monoacylglycerol, fatty acid, platelet activating factor, or dimethylphosphatidyl ethanolamine.

Embodiment 10. The use of any one of the preceding embodiments, whereinsaid extract is selected from lipids shown in Table 1 or comprises anyone or more lipids selected from Table 1.

Embodiment 11. The use of any one of the preceding embodiments, whereinsaid extract comprises any one of the lipids shown in Table 1 as No. 41,No. 71, No. 11, No. 12, No. 38, No. 64, No. 40, No. 37, No. 39, No. 60,No. 62, or its combination with any one or more of the other lipids inTable 1, or its combination with any one or more lipids and otherrelated chemicals.

Embodiment 12. Use of a combination comprising any one or more lipidsshown in Table 1 in the manufacture of a nucleic acid delivery reagent,wherein preferably, the combination comprises any one of the lipidsshown in Table 1 as No. 41, No. 71, No. 11, No. 12, No. 38, No. 64, No.40, No. 37, No. 39, No. 60 and No. 62, or its combination with any oneor more of the other lipids in Table 1, or its combination with any oneor more lipids and other related chemicals, preferably the said nucleicacid is a small nucleic acid, preferably is single or double stranded,preferably the small nucleic acid has a length of 14-32 bp, 16-28 bp or18-24 bp, preferably the reagent is an oral reagent, preferably thenucleic acid is used for treating a disease, such as cancer, for examplegastric cancer or lung cancer.

Embodiment 13. Use of a traditional Chinese medicine in the manufactureof a nucleic acid delivery reagent, wherein preferably the nucleic acidis a small nucleic acid, preferably is single or double stranded,preferably the small nucleic acid has a length of 14-32 bp, 16-28 bp or18-24 bp, preferably the reagent is an oral reagent, preferably thenucleic acid is used for treating a disease, such as cancer, for examplegastric cancer or lung cancer.

Embodiment 14. The use of embodiment 13, wherein said traditionalChinese medicine is selected from Rhodiola crenulata, Taraxacummongolicum, Andrographis paniculata and Lonicera japonica Chinesemedicine decoction pieces.

Embodiment 15. The use of embodiment 13 or 14, wherein the reagentcomprises a compound extracted from a traditional Chinese medicine orartificially synthesized, and preferably, the compound is obtained byextracting a lipid-soluble component by the Bligh & Dyer method, orextracting by decoction of traditional Chinese medicine, morepreferably, the Chinese medicine decoction pieces are soaked in water,and then performed to intense heating and slow heating, and the heatedChinese medicine soup is concentrated, and then is sequentially addedwith chloroform-methanol, chloroform and water for stirring, and thechloroform layer is obtained.

Embodiment 16. The use of embodiment 15, wherein the said compound hasthe structure shown in any one of embodiments 1 to 11, or the reagentcomprises any one or more lipids shown in Table 1, preferably any one oflipids shown in Table 1 as No. 41, No. 71, No. 11, No. 12, No. 38, No.64, No. 40, No. 37, No. 39, No. 60 and No. 62, or its combination withany one or more of the other lipids in Table 1, or its combination withany one or more lipids and other related chemicals.

Embodiment 17. The use of embodiment 16, wherein the compound isselected from the group consisting of lysolecithin, ceramide,diglyceride, phosphatidylethanolamine, phosphatidylcholine,triglyceride, monogalactosyldiglyceride, (neural) sphingosine,phosphatidyl ethanol, monoacylglycerol, fatty acid, platelet activatingfactor, or dimethyl phosphatidyl ethanolamine.

Embodiment 18. The use of embodiment 17, wherein the compound isselected from Table 1.

Embodiment 19. The use of embodiment 18, wherein the said compound isselected from lipids shown in Table 1 as No. 41, No. 71, No. 11, No. 12,No. 38, No. 64, No. 40, No. 37, No. 39, No. 60 and No. 62.

Embodiment 20. The use of any one of embodiments 13-18, wherein thedelivery comprises in vitro cell delivery, or in vivo gastrointestinaldelivery.

Embodiment 21. The use of any one of embodiments 13-20, wherein the useincludes the manufacture of lipid nucleic acid mixture.

Embodiment 22. The use of embodiment 21, wherein the lipid nucleic acidmixture is manufactured by a boiling method, or by a reverse evaporationmethod, or by direct mixing.

Embodiment 23. The use of embodiment 22, wherein temperature in theboiling method is from about 4° C. to about 100° C., from about 25° C.to about 100° C., preferably from about 80° C. to about 100° C., i.e. 4°C., 37° C., 60° C., 80° C. or 100° C.; temperature in the reverseevaporation method is from about 25° C. to about 70° C., preferablyabout 55° C.

Embodiment 24. A pharmaceutical composition comprising one or more lipidextracts of any structure of embodiments 1-11 and a nucleic acid,preferably the lipid is selected from any one or more lipids in Table 1,preferably any one lipid shown in Table 1 as No. 41, No. 71, No. 11, No.12, No. 38, No. 64, No. 40, No. 37, No. 39, No. 60 and No. 62, or itscombination with any one or more of the other lipids in Table 1, or itscombination with any one or more lipids and other related chemicals,wherein preferably the nucleic acid is a small nucleic acid, preferablyis single or double stranded, preferably the small nucleic acid has alength of 14-32 bp, 16-28 bp or 18-24 bp, preferably, the pharmaceuticalcomposition is an oral pharmaceutical combination, preferably thepharmaceutical composition is used for treating a disease, such ascancer, for example gastric cancer or lung cancer.

Embodiment 25. The pharmaceutical composition of embodiment 24, whereinat least part of or all of the lipids and the nucleic acids exist in theform of lipid nucleic acid mixture.

Embodiment 26. The pharmaceutical composition of embodiment 25, whereinthe lipid nucleic acid mixture is prepared by a boiling method, or by areverse evaporation method, or by direct mixing.

Embodiment 27. The pharmaceutical composition of embodiment 26, whereintemperature in the boiling method is from about 4° C. to about 100° C.,from 25° C. to about 100° C., preferably from about 80° C. to 100° C.,i.e. 4° C., 37° C., 60° C., 80° C. or 100° C.; temperature in thereverse evaporation method is from about 25° C. to about 70° C.,preferably about 55° C.

Embodiment 28. A kit comprising one or more lipids having the structureof embodiments 1-11, preferably the lipid is selected from any one ormore lipids in Table 1, preferably shown in Table 1 as No. 41, No. 71,No. 11, No. 12, No. 38, No. 64, No. 40, No. 37, No. 39, No. 60, No. 62,or its combination thereof with any one or more of other lipids in Table1, or its combination thereof with any one or more lipids and otherrelated chemicals, nucleic acids, wherein the lipid and nucleic acid areeach independently provided in a first container and a second container,the first container and the second container are the same or different,wherein preferably the nucleic acid is a small nucleic acid, preferablyis single or double stranded, preferably the small nucleic acid has alength of 14-32 bp, 16-28 bp or 18-24 bp; preferably, the kit is an oralkit, preferably the kit is used for treating a disease, such as cancer,for example gastric cancer or lung cancer.

Embodiment 29. The kit of embodiment 28, wherein at least part of or allof said lipid and nucleic acid are prepared into lipid nucleic acidmixture immediately prior to use.

Embodiment 30. The kit of embodiment 29, wherein the preparation methodof lipid nucleic acid mixture is a boiling method, or a reverseevaporation method, or direct mixing.

Embodiment 31. The kit of embodiment 30, wherein temperature in saidboiling method is from about 4° C. to about 100° C., from 25° C. toabout 100° C., preferably from about 80° C. to about 100° C., i.e. 4°C., 37° C., 60° C., 80° C. or 100° C.; temperature in the reverseevaporation method is from about 25° C. to about 70° C., preferablyabout 55° C.

Embodiment 32. A method of delivering a nucleic acid into a target cell,wherein the nucleic acid is provided in a form of a pharmaceuticalcomposition of any one of embodiments 24-27 or the kit of any one ofembodiments 28-31, preferably the nucleic acid is a small nucleic acid,preferably is single or double stranded, preferably the small nucleicacid has a length of 14-32 bp, 16-28 bp or 18-24 bp; preferably, thenucleic acid is used for treating a disease, such as cancer, for examplegastric cancer or lung cancer.

Embodiment 33. A method of delivering a nucleic acid into a subject invivo in need thereof, wherein the nucleic acid provided from thepharmaceutical composition of any one of embodiments 24-27 or the kit ofany one of embodiments 28-31, wherein preferably said nucleic acid is asmall nucleic acid, preferably is single or double stranded, preferablysaid small nucleic acid has a length of 14-32 bp, 16-28 bp or 18-24 bp;preferably said nucleic acid is used for treating a disease such ascancer, for example gastric cancer or lung cancer.

Embodiment 34. The method of embodiment 33, wherein the subject is ahuman or an animal, such as a mammal.

Embodiment 35. The method of any one of embodiments 33-34, wherein thenucleic acid is delivered to blood circulation or a target tissue/cellof the subject in vivo.

Embodiment 36. The method of embodiment 35, wherein the method includesdirectly delivering the pharmaceutical composition of any one ofembodiments 24-27 or the kit of any one of embodiments 28-31 to asubject in need by digestive tract.

Embodiment 37. The pharmaceutical composition of any one of embodiments24-27, or the kit of any one of embodiments 28-31, wherein the nucleicacid and the lipid are prepared for administration and/or injection.

Embodiment 38. The pharmaceutical composition or the kit of embodiment37, wherein the nucleic acid and lipid are prepared for digestiveadministration or respiratory administration.

Embodiment 39. The pharmaceutical composition or the kit of embodiment37 or 38, wherein the nucleic acid and lipid are prepared for oraladministration or inhalation administration.

Embodiment 40. The pharmaceutical composition or the kit of any one ofembodiments 37-39, wherein the nucleic acid is a small RNA.

Embodiment 41. The pharmaceutical composition or the kit of any one ofembodiments 37-40, wherein the nucleic acid has a stem-loop structure.

Embodiment 42. The pharmaceutical composition, or the kit of any one ofembodiments 37-41, wherein the small RNA has a length of 14-32 bp, or18-24 bp, for example, the length is of 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32 bp.

Embodiment 43. A compound extracted from a traditional Chinese medicineor artificially synthesized can be used for nucleic acid delivery,having the following structure:

L1, L2 or L3 is absent, or L1, L2 and L3 are each independently selectedfrom the group consisting of —C(O)O—CH2-, —CH(OH)—, —C(O)—NH—CH2-,—CH2-OC(O)—, —CH2-NH—C(O)—, —C(O)O—, —C(O)NH—, —OC(O)—, —NH—C(O)—,—CH2-,

with the proviso that at most two of L1, L2 and L3 are absent;

with respect to the divalent groups L1, L2, the dash “-” on the leftside is linked to the groups A and B, respectively, and the dash “-” onthe right side is linked to the central carbon atom;

with respect to the divalent group L3, the dash “-” on the left side islinked to the central carbon atom, and the dash “-” on the right side islinked to the group Q;

A, B and Q are each independently selected from the group consisting ofH, —OH, C1-20 alkyl, C1-20 alkenyl, C1-20 heteroalkyl, C1-20heteroalkenyl, —NH2, and —NR3+, R is H or C1-6 alkyl; and

n is an integer of 0, 1, 2, 3 or 4, preferably the compound is an oralcompound; preferably, the nucleic acid is used for treating a disease,such as cancer, for example gastric cancer or lung cancer.

Embodiment 44. The compound of embodiment 43 wherein

L1 is absent, or L1 is selected from the group consisting of —C(O)O—CH2-and —CH(OH)—,

L2 is absent, or L2 is selected from the group consisting of —C(O)O— and—C(O)NH—,

L3 is absent, or L3 is selected from the group consisting of —C(O)O—,—CH2-OC(O)—, —CH2- and

A is selected from the group consisting of H, C1-20 alkyl and C1-20alkenyl;

B is selected from the group consisting of H, —NH2, C1-20 alkyl andC1-20 alkenyl;

Q is selected from the group consisting of H, —OH, C1-20 alkyl and C1-20alkenyl, and —NR3+, wherein R is H or C1-6 alkyl, wherein preferably thetraditional Chinese medicine is selected from Rhodiola crenulata,Taraxacum mongolicum, Andrographis paniculata and Lonicera japonicaChinese medicine decoction pieces, preferably the compound is obtainedby extracting a lipid-soluble component by the Bligh & Dyer method, morepreferably by soaking Chinese medicine decoction pieces in water, andthen sequentially performing intense heating and slow heating, and theheated Chinese medicine soup is concentrated, and then is added withchloroform-methanol, chloroform and water for stirring, and thechloroform layer is obtained; preferably, the nucleic acid is a smallnucleic acid, preferably is single or double-stranded, preferably thesmall nucleic acid has a length of 14-32 bp, 16-28 bp or 18-24 bp.

Embodiment 45. The compound of embodiment 43 or 44, having the followingformula:

Embodiment 46. The compound of any one of embodiments 43-45, wherein

A is selected from the group consisting of H, C10-20 alkyl and C10-20alkenyl;

B is selected from the group consisting of H, —NH2, C10-20 alkyl andC0-20 alkenyl;

Q is selected from the group consisting of H, —OH, C10-20 alkyl andC10-20 alkenyl, and —NR3+ wherein R is H or C1-4 alkyl.

Embodiment 47. The compound of any one of embodiments 43-46, wherein

A is selected from the group consisting of H, a straight-chain C15-18alkyl group and a straight-chain C15-18 alkenyl group;

B is selected from the group consisting of H, —NH2, a straight-chainC15-18 alkyl group and a straight-chain C15-18 alkenyl group;

Q is selected from the group consisting of H, —OH, a straight-chainC15-18 alkyl group and a straight-chain C15-18 alkenyl group, and —NR3+wherein R is H or a C1-4 alkyl group;

the alkenyl group in the A, B, Q has 1-5 double bonds.

Embodiment 48. The compound of any one of embodiments 43-47, wherein inthe A, B, Q of the said structure, the alkenyl group has 1-4 doublebonds, and is in a Z configuration.

Embodiment 49. The compound of any one of embodiments 43-48, wherein inthe A, B, Q of the structure, the alkenyl group has 1-3 double bonds andis in a Z configuration.

Embodiment 50. The compound of any one of embodiments 43-49, wherein thecompound is selected from the following formulas:

wherein

A is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-18 alkenyl group;

B is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-18 alkenyl group;

Q is selected from the group consisting of H, —OH, a straight-chainC15-18 alkyl group and a straight-chain C15-18 alkenyl group, and —NR3+wherein R is H or methyl; and

L3 is —C(O)O—.

Embodiment 51. The compound of any one of embodiments 43-50, wherein thecompound is selected from lipids shown in Table 1.

Embodiment 52. The compound of any one of embodiments 43-51, wherein thecompound is selected from lipids shown in Table 1 as No. 41, No. 71, No.11, No. 12, No. 38, No. 64, No. 40, No. 37, No. 39, No. 60 or No. 62.

Embodiment 53. A method of facilitating nucleic acid delivery comprisingheating or warming up a nucleic acid and a traditional Chinese medicineextract, any compound derived naturally or synthetically, preferably thelipid of any one of embodiments 1 to 11; temperature for heating orwarming up is preferably from about 4° C. to about 100° C., from about25° C. to about 100° C., preferably from about 50° C. to about 100° C.,more preferably from about 95° C. to about 100° C., particularlypreferably from about 80° C. to about 100° C., i.e. 4° C., 37° C., 60°C., 80° C. or 100° C., wherein preferably the nucleic acid is a smallnucleic acid, preferably is single or double stranded, preferably thesmall nucleic acid has a length of 14-32 bp, 16-28 bp or 18-24 bp;preferably, the nucleic acid delivery is by oral administration;preferably, the nucleic acid is used for treating a disease, such ascancer, for example gastric cancer or lung cancer.

Embodiment 54. The method of embodiment 53, wherein the traditionalChinese medicine extract comprises a compound of the structure as setforth in embodiments 1-9.

Embodiment 55. The method of embodiment 53, wherein the traditionalChinese medicine extract comprises any one or more lipids shown in Table1.

Embodiment 56. The method of embodiment 53, wherein the traditionChinese medicine extract comprises any one of lipids shown in Table 1 asNo. 41, No. 71, No. 11, No. 12, No. 38, No. 64, No. 40, No. 37, No. 39,No. 60 and No. 62, or its combination with any one or more of the otherlipids in Table 1, or its combination with any one or more lipids andother related chemicals.

Embodiment 57. The use of embodiment 11, 12 or 16, the pharmaceuticalcomposition of embodiment 24, or the kit of embodiment 28, wherein thecombination is any one of the following: a lipid combination of No.8:No. 41=6:1; a lipid combination of No. 38:No. 41=6:1; a lipidcombination of No. 39:No. 41=6:1; a lipid combination of No. 40:No.41=6:1; a lipid combination of No. 38:No. 12:No. 41:No. 29=1:1:2:1; alipid combination of No. 40:No. 12:No. 41=2:4:3; a lipid combination ofNo. 12:No. 41=1:6; a lipid combination of No. 12:No. 41=1:1; a lipidcombination of No. 12:No. 41=6:1; a lipid combination of No. 40:No.12:No. 41=2:2:2; a lipid combination of No. 4:No. 12:No. 41=1:1:1; DGcombination of No. 1:No. 2:No. 3:No. 19:No. 35=1:1:1:1:1; TG combinationof No. 6:No. 9:No. 10:No. 13:No. 15:No. 16:No. 18:No. 20:No. 21:No.22:No. 23:No. 24:No. 25:No. 26:No. 27:No. 28:No. 32:No.33=1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1; LPC combination of No. 36:No.37=1:1; PC combination of No. 11:No. 12=1:1; PE combination of No. 8:No.38=1:1; Cer combination of No. 4:No. 14=1:1; So combination of No.17:No. 30:No. 31=1:1:1; an equal volume combination of No. 1-36 withoutNo. 5, No. 7; an equal volume combination of No. 1-36 without No. 5, No.7, No. 34; an equal volume combination of No. 1-36 without No. 5, No. 7,No. 1, No. 2, No. 3, No. 19, No. 35; an equal volume combination of No.1-36 No. 5, No. 7, No. 6, No. 9, No. 10, No. 13, No. 15, No. 16, No. 18,No. 20, No. 21, No. 22, No. 23, No. 24, No. 25, No. 26, No. 27, No. 28,No. 32, No. 33; an equal volume combination of No. 1-36 without No. 5,No. 7, No. 36, No. 37; an equal volume combination of No. 1-36 withoutNo. 5, No. 7, No. 11, No. 12; an equal volume combination of No. 1-36without No. 5, No. 7, No. 8 in; an equal volume combination of No. 1-36without No. 5, No. 7, No. 4, No. 14; an equal volume combination of No.1-36 without No. 5, No. 7, No. 29; a lipid combination of No. 1:No.34=2:1; a lipid combination of No. 1: said DG composition=2:1; a lipidcombination of No. 1: said TG composition=2:1; a lipid combination ofNo. 1: said LPC composition=2:1; a lipid combination of No. 1:No. 8=2:1;a lipid combination of No. 1:No. 12=2:1; a lipid combination of No. 1:said Cer composition=2:1; a lipid combination of No. 1: said Socomposition=2:1; a lipid combination of No. 1:No. 29=2:1; a lipidcombination of No. 1:No. 8:No. 12=1:1:1; a lipid combination of No.8:No. 34=2:1; a lipid combination of No. 8: said DG composition=2:1; alipid combination of No. 8: said TG composition=2:1; a lipid combinationof No. 8: said LPC composition=2:1; a lipid combination of No. 8:No.37=4:1; a lipid combination of No. 8:No. 12=2:1; a lipid combination ofNo. 8: said Cer composition=2:1; a lipid combination of No. 8: said Socomposition=2:1; a lipid combination of No. 8:No. 31=6:1; a lipidcombination of No. 8:No. 29=2:1; a lipid combination of No. 12:No.34=2:1; a lipid combination of No. 12: said DG composition=2:1; a lipidcombination of No. 12: said TG composition=2:1; a lipid combination ofNo. 12: said LPC composition=2:1; a lipid combination of No. 12:No.8=2:1; a lipid combination of No. 12: said Cer composition=2:1; a lipidcombination of No. 12: said So composition=2:1; a lipid combination ofNo. 12:No. 29=2:1; a lipid combination of No. 12:No. 8:No. 1&2=2:1:1; alipid combination of No. 12:No. 8:No. 15=2:1:1; a lipid combination ofNo. 12:No. 8:No. 36&37=2:1:1; a lipid combination of No. 12:No. 8:No.11=2:1:1; a lipid combination of No. 12:No. 8:No. 12=2:1:1; a lipidcombination of No. 12:No. 8:No. 4=2:1:1; a lipid combination of No.12:No. 8:No. 31=2:1:1; a lipid combination of No. 12:No. 8:No. 29=2:1:1;a lipid combination of No. 12:No. 8:No. 34=3:2:1; a lipid combination ofNo. 12:No. 8:No. 34=4:2:3; a lipid combination of No. 12:No. 8:No.2=4:2:3; a lipid combination of No. 12:No. 8:No. 2=16:8:3; a lipidcombination of No. 12:No. 8:No. 32=4:2:3; a lipid combination of No.12:No. 8:No. 37=4:2:3; a lipid combination of No. 12:No. 8:No. 11=4:2:3;a lipid combination of No. 12:No. 8:No. 38=4:2:3; a lipid combination ofNo. 12:No. 8:No. 4=4:2:3; a lipid combination of No. 12:No. 8:No.31=4:2:3; a lipid combination of No. 12:No. 8:No. 29=4:2:3; a lipidcombination of No. 12:No. 8:No. 29:No. 31=2:1:1:1; a lipid combinationof No. 12:No. 8:No. 29:No. 31:No. 34=4:2:2:2:5; a lipid combination ofNo. 12:No. 8:No. 29:No. 31:No. 2=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 32=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 11=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 37=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 38=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 4=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 4:No. 1:No. 16=2:1:1:3:2:2:3; a lipidcombination of No. 1:No. 8:No. 12:No. 1&2=2:2:2:3; a lipid combinationof No. 1:No. 8:No. 12:No. 15=2:2:2:3; a lipid combination of No. 1:No.8:No. 12:No. 36&37=2:2:2:3; a lipid combination of No. 1:No. 8:No.12:No. 12=2:2:2:3; a lipid combination of No. 1:No. 8:No. 12:No.4=2:2:2:3; a lipid combination of No. 1:No. 8:No. 12:No. 31=2:2:2:3; alipid combination of No. 1:No. 8:No. 12:No. 29=2:2:2:3; a lipidcombination of No. 8:No. 34:No. 1&2=2:1:1; a lipid combination of No.8:No. 34:No. 15=2:1:1; a lipid combination of No. 8:No. 34:No.36&37=2:1:1; a lipid combination of No. 8:No. 34:No. 12=2:1:1; a lipidcombination of No. 8:No. 34:No. 4=2:1:1; a lipid combination of No.8:No. 34:No. 31=2:1:1; a lipid combination of No. 8:No. 34:No. 29=2:1:1;a lipid combination of No. 8:No. 31:No. 34=12:3:5; a lipid combinationof No. 8:No. 31:No. 2=12:3:5; a lipid combination of No. 8:No. 31:No.37=12:3:5; a lipid combination of No. 8:No. 31:No. 11=12:3:5; a lipidcombination of No. 8:No. 31:No. 12=12:3:5; a lipid combination of No.8:No. 31:No. 4=12:3:5; a lipid combination of No. 8:No. 31:No.29=12:3:5; a lipid combination of No. 8:No. 31:No. 32=12:3:5; a lipidcombination of No. 8:No. 4:No. 34=12:3:5; a lipid combination of No.8:No. 4:No. 2=12:3:5; a lipid combination of No 0.8:No. 4:No. 37=12:3:5;a lipid combination of No. 8:No. 4:No. 12=12:3:5; a lipid combination ofNo. 8:No. 4:No. 31=12:3:5; a lipid combination of No. 8:No. 4:No.29=12:3:5; a lipid combination of No. 8:No. 4:No. 32=12:3:5; a lipidcombination of No. 38:No. 34=2:1; a lipid combination of No. 38:No.1=2:1; a lipid combination of No. 38:No. 2=2:1; a lipid combination ofNo. 38:No. 1&2=2:1; a lipid combination of No. 38:No. 15=2:1; a lipidcombination of No. 38:No. 32=2:1; a lipid combination of No. 38:No.37=2:1; a lipid combination of No. 38:No. 37=4:1; a lipid combination ofNo. 38:No. 11=2:1; a lipid combination of No. 38:No. 12=2:1; a lipidcombination of No. 38:No. 11&12=2:1; a lipid combination of No. 38:No.12=4:1; a lipid combination of No. 38:No. 8=2:1; a lipid combination ofNo. 38:No. 4=2:1; a lipid combination of No. 38: So (30)=2:1; a lipidcombination of No. 38:No. 31=2:1; a lipid combination of No. 38:No.29=2:1; a lipid combination of No. 1:No. 38:No. 12:No. 34=2:2:2:3; alipid combination of No. 1:No. 38:No. 12:No. 15=2:2:2:3; a lipidcombination of No. 1:No. 38:No. 12:No. 37=2:2:2:3; a lipid combinationof No. 1:No. 38:No. 12:No. 8=2:2:2:3; a lipid combination of No. 1:No.38:No. 12:No. 4=2:2:2:3; a lipid combination of No. 1:No. 38:No. 12:No.31=2:2:2:3; a lipid combination of No. 1:No. 38:No. 12:No. 29=2:2:2:3; alipid combination of No. 38:No. 34:No. 1=2:1:3; a lipid combination ofNo. 38:No. 34:No. 15=2:1:3; a lipid combination of No. 38:No. 34:No.37=2:1:3; a lipid combination of No. 38:No. 34:No. 12=2:1:3; a lipidcombination of No. 38:No. 34:No. 8=2:1:3; a lipid combination of No.38:No. 34:No. 4=2:1:3; a lipid combination of No. 38:No. 34:No.31=2:1:3; a lipid combination of No. 38:No. 34:No. 29=2:1:3; a lipidcombination of No. 38:No. 12:No. 1=2:1:3; a lipid combination of No.38:No. 12:No. 2=4:1:3; a lipid combination of No. 38:No. 12:No.15=2:1:3; a lipid combination of No. 38:No. 12:No. 37=2:1:3; a lipidcombination of No. 38:No. 12:No. 8=2:1:3; a lipid combination of No.38:No. 12:No. 4=2:1:3; a lipid combination of No. 38:No. 12:No.31=2:1:3; a lipid combination of No. 38:No. 12:No. 29=2:1:3; a lipidcombination of No. 38:No. 12:No. 1:No. 15:No. 34=22:22:22:33:36; a lipidcombination of No. 38:No. 12:No. 1:No. 15:No. 37=22:22:22:33:36; a lipidcombination of No. 38:No. 12:No. 1:No. 15:No. 4=22:22:22:33:36; a lipidcombination of No. 38:No. 12:No. 1:No. 15:No. 31=22:22:22:33:36; a lipidcombination of No. 38:No. 12:No. 1:No. 15:No. 29=22:22:22:33:36; a lipidcombination of No. 38:No. 34:No. 37:No. 1=44:22:33:36; a lipidcombination of No. 38:No. 34:No. 37:No. 15=44:22:33:36; a lipidcombination of No. 38:No. 34:No. 37:No. 12=44:22:33:36; a lipidcombination of No. 38:No. 34:No. 37:No. 4=44:22:33:36; a lipidcombination of No. 38:No. 34:No. 37:No. 31=44:22:33:36; a lipidcombination of No. 38:No. 12:No. 4:No. 34=44:22:33:36; a lipidcombination of No. 38:No. 12:No. 4:No. 1=44:22:33:36; a lipidcombination of No. 38:No. 12:No. 4:No. 15=44:22:33:36; a lipidcombination of No. 38:No. 12:No. 4:No. 37=44:22:33:36; a lipidcombination of No. 38:No. 12:No. 4:No. 37=8:2:5:3; a lipid combinationof No. 38:No. 12:No. 4:No. 31=44:22:33:36; a lipid combination of No.38:No. 12:No. 4:No. 29=44:22:33:36; a lipid combination of No. 38:No.12:No. 4:No. 29:No. 34=88:44:66:72:135; a lipid combination of No.38:No. 12:No. 4:No. 29:No. 1=88:44:66:72:135; a lipid combination of No.38:No. 12:No. 4:No. 29:No. 15=88:44:66:72:135; a lipid combination ofNo. 38:No. 12:No. 4:No. 29:No. 37=88:44:66:72:135; a lipid combinationof No. 38:No. 12:No. 4:No. 29:No. 31=88:44:66:72:135; a lipidcombination of No. 38:No. 12:No. 4:No. 2=20:10:15:9; a lipid combinationof No. 38:No. 12:No. 4:No. 6=20:10:15:9; a lipid combination of No.38:No. 12:No. 4:No. 17=20:10:15:9; a lipid combination of No. 38:No.12:No. 4:No. 29=20:10:15:9; a lipid combination of No. 38:No. 12:No.4:No. 34=20:10:15:9; a lipid combination of No. 38:No. 12:No. 4:No.37=20:10:15:9; a lipid combination of No. 38:No. 12:No. 31:No.34=2:1:3:3; a lipid combination of No. 38:No. 12:No. 31:No. 1=2:1:3:3; alipid combination of No. 38:No. 12:No. 31:No. 15=2:1:3:3; a lipidcombination of No. 38:No. 12:No. 31:No. 37=2:1:3:3; a lipid combinationof No. 38:No. 12:No. 31:No. 4=2:1:3:3; a lipid combination of No. 38:No.12:No. 31:No. 29=2:1:3:3; a lipid combination of No. 38:No. 34:No.37:No. 31:No. 1=88:44:66:72:135; a lipid combination of No. 38:No.34:No. 37:No. 31:No. 15=88:44:66:72:135; a lipid combination of No.38:No. 34:No. 37:No. 31:No. 12=88:44:66:72:135; a lipid combination ofNo. 38:No. 34:No. 37:No. 31:No. 4=88:44:66:72:135; a lipid combinationof No. 38:No. 34:No. 37:No. 31:No. 29=88:44:66:72:135; a lipidcombination of No. 38:No. 37:No. 34=4:2:3; a lipid combination of No.38:No. 37:No. 1=4:2:3; a lipid combination of No. 38:No. 37:No. 2=4:2:3;a lipid combination of No. 38:No. 37:No. 1&2=4:2:3; a lipid combinationof No. 38:No. 37:No. 2=32:8:5; a lipid combination of No. 38:No. 37:No.32=32:8:5; a lipid combination of No. 38:No. 37:No. 15=4:2:3; a lipidcombination of No. 38:No. 37:No. 32=4:2:3; a lipid combination of No.38:No. 37:No. 8=4:2:3; a lipid combination of No. 38:No. 37:No.11=4:2:3; a lipid combination of No. 38:No. 37:No. 12=4:2:3; a lipidcombination of No. 38:No. 37:No. 11&12=4:2:3; a lipid combination of No.38:No. 37:No. 12=4:1:1; a lipid combination of No. 38:No. 37:No.4=4:2:3; a lipid combination of No. 38:No. 37:No. 30=4:2:3; a lipidcombination of No. 38:No. 37:No. 31=4:2:3; a lipid combination of No.38:No. 37:No. 29=4:2:3; a lipid combination of No. 8:No. 37:No.32=4:1:2; a lipid combination of No. 8:No. 37:No. 2=4:1:2; a lipidcombination of No. 38:No. 37:No. 15:No. 34=64:16:10:45; a lipidcombination of No. 38:No. 37:No. 15:No. 1=64:16:10:45; a lipidcombination of No. 38:No. 37:No. 15:No. 12=64:16:10:45; a lipidcombination of No. 38:No. 37:No. 15:No. 4=64:16:10:45; a lipidcombination of No. 38:No. 37:No. 15:No. 31=64:16:10:45; a lipidcombination of No. 38:No. 37:No. 15:No. 29=64:16:10:45; a lipidcombination of No. 38:No. 2:No. 37=4:2:3; a lipid combination of No.38:No. 2:No. 31=4:2:3; a lipid combination of No. 38:No. 2:No. 29=4:2:3;a lipid combination of No. 38:No. 2:No. 34=4:2:3; a lipid combination ofNo. 38:No. 2:No. 32=4:2:3; a lipid combination of No. 38:No. 2:No.12=4:2:3; a lipid combination of No. 38:No. 2:No. 12=4:5:1; a lipidcombination of No. 38:No. 2:No. 4=4:2:3; No. 1&2, No. 11&12 and No.36&37 represent lipids No. 1 and No. 2 in any ratio, lipids No. 11 andNo. 12 in any ratio, lipids No. 36 and No. 37 in any ratio,respectively.

Embodiment 58. Use of a compound having the following formula for themanufacture of nucleic acid delivery reagent:

wherein

L1, L2 or L3 is absent, or L1, L2 and L3 are each independently selectedfrom the group consisting of —C(O)O—CH2-, —CH(OH)—, —CH2-OC(O), —C(O)O—,—C(O)NH—;

with the proviso that at most two of L1, L2 and L3 are absent;

with respect to the divalent groups L1, L2, the dash “-” on the leftside is linked to the groups A and B, respectively, and the dash “-” onthe right side is linked to the central carbon atom;

with respect to the divalent group L3, the dash “-” on the left side islinked to the central carbon atom, and the dash “-” on the right side islinked to the group Q;

A, B and Q are independently selected from the group consisting of H,—OH, C1-20 alkyl, C1-20 alkenyl, —NH2, and —NR3+, R is H or C1-6 alkyl;preferably the reagent is an oral reagent; preferably, the nucleic acidis used for treating a disease, such as cancer, for example gastriccancer or lung cancer.

Embodiment 59. The use of embodiment 58, wherein the compound has thefollowing structure:

wherein

A is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-20 alkenyl group;

B is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-20 alkenyl group;

Q is —OH;

preferably,

A is selected from the group consisting of a straight-chain C15-20 alkylgroup and a straight-chain C15-20 alkenyl group;

B is selected from the group consisting of a straight-chain C15-20 alkylgroup and a straight-chain C15-20 alkenyl group;

Q is —OH;

preferably,

A is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-18 alkenyl group;

B is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-18 alkenyl group;

Q is —OH.

Embodiment 60. The use of embodiment 58, wherein the compound has thefollowing structure:

wherein

A is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-22 alkenyl group;

B is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-22 alkenyl group;

Q is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-22 alkenyl group;

preferably,

A is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-22 alkenyl group;

B is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-22 alkenyl group;

Q is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-22 alkenyl group;

preferably,

A is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-20 alkenyl group;

B is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-20 alkenyl group;

Q is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-20 alkenyl group.

Embodiment 61. The use of embodiment 58, wherein the compound has thefollowing structure:

wherein

A is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-20 alkenyl group;

B is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-20 alkenyl group;

Q is —OH;

preferably,

A is selected from the group consisting of a straight-chain C15-20 alkylgroup and a straight-chain C15-18 alkenyl group;

B is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-18 alkenyl group;

Q is —OH;

preferably,

A is a straight-chain C15-20 alkyl group;

B is a straight-chain C15-18 alkyl group;

Q is —OH.

Embodiment 62. The use of embodiment 58, wherein the compound has thefollowing structure:

wherein

A is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-20 alkenyl group;

Q is —OH;

preferably,

A is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C15-18 alkenyl group;

Q is —OH;

preferably,

A is a straight-chain C15-20 alkyl group;

Q is —OH.

Embodiment 63. The use of any one of embodiments 1-23, thepharmaceutical composition of any one of embodiments 24-27, the kit ofany one of embodiments 28-31, the method of any one of embodiments 32-36and 53-56, or the method of embodiment 43, wherein the lipid or compoundhas the following structure:

wherein

A is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-20 alkenyl group;

B is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-20 alkenyl group;

Q is —OH;

preferably,

A is selected from the group consisting of a straight-chain C15-20 alkylgroup and a straight-chain C15-20 alkenyl group;

B is selected from the group consisting of a straight-chain C15-20 alkylgroup and a straight-chain C15-20 alkenyl group;

Q is —OH;

preferably,

A is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-18 alkenyl group;

B is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-18 alkenyl group;

Q is —OH.

Embodiment 64. The use of embodiment 63, the pharmaceutical composition,the kit, or the method, wherein said compound has the followingstructure:

wherein

A is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-22 alkenyl group;

B is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-22 alkenyl group;

Q is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-22 alkenyl group;

preferably,

A is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-22 alkenyl group;

B is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-22 alkenyl group;

Q is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-22 alkenyl group;

preferably,

A is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-20 alkenyl group;

B is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-20 alkenyl group;

Q is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-20 alkenyl group.

Embodiment 65. The use of embodiment 63, the pharmaceutical composition,the kit, or the method, wherein the compound has the followingstructure:

wherein

A is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-20 alkenyl group;

B is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-20 alkenyl group;

Q is —OH;

preferably,

A is selected from the group consisting of a straight-chain C15-20 alkylgroup and a straight-chain C15-18 alkenyl group;

B is selected from the group consisting of a straight-chain C15-18 alkylgroup and a straight-chain C15-18 alkenyl group;

Q is —OH;

preferably,

A is a straight-chain C15-20 alkyl group;

B is a straight-chain C15-18 alkyl group;

Q is —OH.

Embodiment 66. The use of embodiment 63, the pharmaceutical composition,the kit, or the method, wherein the compound has the followingstructure:

wherein

A is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C10-20 alkenyl group;

Q is —OH;

preferably,

A is selected from the group consisting of a straight-chain C10-20 alkylgroup and a straight-chain C15-18 alkenyl group;

Q is —OH;

preferably,

A is a straight-chain C15-20 alkyl group;

Q is —OH.

EXAMPLES

The following examples are merely illustrative of the inventiondisclosed herein, and are not to be construed as limiting the scope ofthe appended claims.

TABLE 2 Small RNA and their sequences used in the examples SEQ ID NosiRNA Sequence Length 1 sly-miR168b- TCGCTTGGTGCAGGTCGGGAC 21 5p 2Pab-miR3711 GGCCCTCCTTCTAGCGCCA 19 3 CXL-sRNA-17 CAGAGTCGCGCAGCGGAA 18 4PGY-sRNA-6 GTTCAGAGTTCTACAGTCCGA 21 5 PGY-sRNA-18CGGGGCTACGCCTGTCTGAGCGTCGC 26 6 HJT-sRNA-m7 TGAGGTAGTAGGTTGTGTGGTTGTAAGC28 7 HJT-sRNA-3 CAGCCAAGGATGACTTGCCGG 21 8 HJT-sRNA-a2TAGCACCATCCGAAATCGGTA 21 9 HJT-sRNA-h3 TGGGGCTACGCCTGTCTGAGCGTCGCT 27 10si-XRN2 GAGUACAGAUGAUCAUGUUGAGTACAG 19 ATGATCATGTT 11 si-Ssu72GACUCACGUGAAGCUUCCAGACTCACG 19 TGAAGCTTCCA 12 si-CPSF4GAGUCAUCUGUGUGAAUUAGAGTCATCT 19 GTGTGAATTA 13 si-LAMP1CAAUGCGAGCUCCAAAGAA 19 14 si-LAMP2 GCGGUCUUAUGCAUUGGAA 19 15 si-NFκBAGUACCCUGAAGCUAUAUUUU 21 16 si-TNFα CACAACCAACUAGUGGUGCUU 21 17PGY-sRNA-23 CCCTCCGCGGCCAGCTTCT 19 18 PGY-sRNA-26TCCGGAATGATTGGGCGTAAAGCGT 25 19 PGY-sRNA-32 CCGGCCCCGAACCCGTCGGC 20Note: Double-stranded sRNA is indicated by the “si-” prefix.

Examples for Lipids Shown in Table 1 as No. 1-32

1. Extraction of Lipids from Traditional Chinese Medicine

1.1 Decoction of Traditional Chinese Medicine

1) 100 g Chinese medicine decoction pieces (Rhodiola crenulata,purchased from Ningbo Haishu Qiancao Biotechnology Co., Ltd.; Taraxacummongolicum, Lonicera japonica, Andrographis paniculata, purchased fromBeijing Tongrentang pharmacy), was added into 1000 mL ddH₂O and soakedfor 30 min.

2) The Chinese medicine decoction pot was boiled for 15 min with intenseheating, and for 20 min with slow heating.

3) 400 mL of the heated Chinese medicine soup was added to a rotaryevaporator at 60° C., 60 rpm, and concentrated to 100 mL.

1.2 Lipid Extraction

1) Chloroform-methanol mixture (chloroform:methanol=1:2, v/v) 600 ml wasadded to the Chinese medicine soup obtained from the above step 1.1(concentrated by rotary evaporator), to makechloroform:methanol:water=1:2:0.8, and stirred for 10-15 min to mix.

2) 200 mL chloroform was added to an Erlenmeyer flask, and stirred for10 min to mix.

3) 200 ml ddH₂O was added to the Erlenmeyer flask to makechloroform:methanotwater=2:2:1.8, and stirred for 10 min to mix.

4) The liquid of upper layer and the insoluble substances fromintermediate layer were removed, and the lower chloroform layer wereobtained. Storage at −40° C.

1.3 HPLC-MS/MS Identification of Lipid Components

Instrument Setup

1) Chromatography Setup:

Instrument: Ultimate 3000; column: Kinetex C18 (100×2.1 mm, 1.9 μm);column temperature: 45° C.; mobile phase A: acetonitrile:water (V/V,60:40), the solution containing 10 mmol/L ammonium formate, mobile phaseB: acetonitrile:isopropanol (10:90, V/V), the solution containing 10mmol/L ammonium formate and 0.1% formic acid. Flow rate: 0.4 mL/min;injection volume: 4 μl.

2) Mass Spectrometry Parameters:

a) Positive mode: Heater Temp 300° C., Sheath Gas Flow rate, 45 arb, AuxGas Flow Rate, 15 arb, Sweep Gas Flow Rate, 1 arb, spray volt age, 3.0KV, Capillary Temp, 350° C., S-Lens RF Level, 30%. Scan ranges:200-1500.

b) Negative mode: Heater Temp 300° C., Sheath Gas Flow rate, 45 arb, AuxGas Flow Rate, 15 arb, Sweep Gas Flow Rate, 1 arb, spray voltage, 2.5KV, Capillary Temp, 350° C., S-Lens RF Level, 60%. Scan ranges:200-1500.

1.4 Identification of the Lipids Derived from the Chinese Medicines

The lipid components were identified by HPLC-MS/MS, and a total of 138lipid components derived from traditional Chinese medicines wereidentified, among which 125 were identified in positive mode and 13 innegative mode. The following experiments were performed on compounds No.1-32 as shown in Table 1.

It should be noted that the lipids tested below were all commerciallypurchased or commercially synthesized, and used as described in Table1-1.

2. Manufacture of Lipid Nucleic Acid Mixture

2.1 Reverse Evaporation Method:

600 μl lipid in diethyl ether solution was prepared, and groupedaccording to the lipid number shown in Table 1, wherein the diethylether solution had a concentration of 0.017857 mg/mL for the lipid groupNo. 1/2/4/9/14/18/19/20/21/22/23/24/25/26/27/28/29/30/32, 0.035714 mg/mLfor the lipid group No. 3/8/10/11/12/13, and 0.0035714 mg/mL for thelipid group No. 6/15/16/17/31; the lipid solution was added to 120 μlHJT-sRNA-m7 single-stranded RNA in DEPC-treated aqueous solution (15nmol) in a volume ratio of 5:1, and sonitcated for 3 min. Diethyl etherwas removed by evaporation at 55° C., and then 600 DEPC water was addedfor hydration to give HJT-sRNA-m7 lipid mixture.

2.2 Boiling Method:

60 μl lipid in chloroform solution was prepared, and grouped accordingto the lipid numbers shown in Table 1, wherein the chloroform solutionhad a concentration of 5 mg/mL for the lipid group No.1/2/4/9/14/18/19/20/21/22/23/24/25/26/27/28/29/30/32, 10 mg/mL for thelipid group No. 3/8/10/11/12/13, 1 mg/mL for the lipid groupNo.6/15/16/17/32; the above lipid chloroform solution was mixed with 600 μlHJT-sRNA-m7 single-stranded RNA in DEPC-treated aqueous solution (15nmol) and heated at 100° C. for 30 min to give HJT-sRNA-m7 lipidmixture.

3. In Vitro Delivery Experiment of Lipid Nucleic Acid Mixture

3.1 NCI-N87 cell (gastric cancer cell), MRC-5 cell (lung fibroblast),A549 cell (lung cancer cell) were cultured to logarithmic growth phase,and then plated to a six-well plate at cell density of 1×10⁶/2 mLmedium/well; MRC-5 cell was cultured in Eagle's MEM medium (MEM, Gibco);A549 cell was cultured in Ham's F-12 medium (HyClone); NCI-N87 cell wascultured in RPMI-1640 medium (HyClone); followed by incubation overnightat 37° C., and the follow-up experiments were performed after the cellswere attached to the walls.

3.2 Experimental Groups as Follows:

1) NC group: referred to untreated cells; this group served as anegative control group.

2) RNAimax treatment group: 2 μl RNAimax transfection reagent andHJT-sRNA-m7 solution were diluted in 100 μl opti-MEM medium respectivelyand then the two were mixed, allowed to stand for 15 min, added intocells and then mixed. The final concentration of HJT-sRNA-m7 was 200 nM;this group served as a positive control group.

3) Free uptake group: HJT-sRNA-m7 solution was directly added (the finalconcentration was 200 nM), and the group served as a negative controlgroup.

4) Lipid nucleic acid mixture: the mixture of lipid and HJT-sRNA-m7prepared from the step 2 were added into cells and mixed, and the finalconcentration of RNA was kept at 200 nM.

3.3 After co-incubation with the small RNA for 3 hours, the cells werewashed 2-3 times with PBS. The cells were harvested with TRIzol lysisbuffer, and the total RNA was extracted. The abundance of small RNA thatentered cells was detected by RT-qPCR, and the localization of RNA wasdetected by fluorescence in situ hybridization; protocols of eachdetection method were as follows:

3.3.1 RT-qPCR Detection of Small RNA (Taqman Probe Method)

1) The sRNA was reverse transcribed to cDNA: Reverse Transcription Kit(TaqMan® MicroRNA Reverse Transcription Kit, cat. No. 4366597) was usedto reverse transcribe sRNA into cDNA.The reverse transcription systemwas as follows: 100 mM dNTPs (with dTTP) 0.15 μl, MultiScribe™ reversetranscriptase 50 U/μl 1.00 μl, 10×RT buffer 1.5 RNase inhibitor (20U/μ1) 0.19 μl, nuclease-free H₂O 4.6 μl, 5 μl RNA template (200 ng/μl)was added after mixing, 3 μl 5× Taqman probe primer was added aftermixing, brief centrifuging after mixing, and then kept on ice for 5 minbefore loading into a PCR reactor. The reaction condition was asfollows: (1) 16° C., 30 min; (2) 42° C., 30 min; (3) 85° C., 5 min; (4)4° C., termination of reaction. 10 μl RNase-free ddH₂O was added to makeup the final volume to 25 μl after the reaction. The Taqman probe primerused in the reverse transcription process was synthesized by Invitrogen(U6: 4440887, HJT-sRNA-m7: 4398987).

2) Quantitative PCR amplification reaction: qPCR reaction system had atotal volume of 10 μl, containing: 5 μl 2×TaqMan® Universal Master MixII, with UNG, 0.5 μl 20× Taqman Primer, 1 μl cDNA by reversetranscription, 3.5 μl RNase-free dH₂O. LightCycler 480 fluorescencequantitative PCR instrument was used, and the PCR reaction conditionswere: 50° C. for 2 min, 95° C. for 10 min for pre-denaturation, followedby PCR amplification cycle: (1) 95° C., 15 s; (2) 60° C., 60 s; (3) 60°C., 60 s; a total of 40 cycles; 40° C. for 10 s in the end to cool down.The Taqman probe for the amplification reaction was designed andsynthesized by Invitrogen (U6: 4440887, HJT-sRNA-m7: 4398987).

3) The relative expression level was calculated by 2-ΔCt method.

3.3.2 RT-qPCR Detection of Small RNA (SYBR Green Dye Method)

1) The sRNA was reverse transcribed to cDNA: Reverse Transcription Kit(High-Capacity cDNA Reverse Transcription Kits, Applied Biosystems, cat.No. 4368813) was used to reverse transcribe sRNA into cDNA by stem-loopmethod, and the reverse transcription system was as follows: RNAtemplate (150 ng/μl) 10 μl, 10×RT buffer, 2.0 μl, 25×dNTP Mix (100 mM)0.8 μl, U6 RT stem-loop primer 2.0 μl, HJT-sRNA-RT-m7 stem-loop primer2.0 μl, MultiScribe™ reverse transcriptase 1.0 μl, RNase inhibitor 1.0μl, nuclease-free H₂O 1.2 μl, loaded into a PCR reactor after briefcentrifugation, the reaction conditions were as follows: (1) 25° C., 10min; (2) 37° C., 120 min; (3) 85° C., 5 min; (4) 4° C., termination ofthe reaction. 20 μl RNase-free ddH₂O was added to make up the finalvolume to 40 μl after the reaction. The stem-loop primer used in thereverse transcription process was synthesized by Beijing TsingkeBiotechnology Co., Ltd. (U6 RT primer:GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAAAT ATG (SEQ ID NO: 21);HJT-sRNA-m7 RT stem-loop primer:GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACGCTTAC AA (SEQ ID NO: 22)).

2) Quantitative PCR amplification reaction: the qPCR reaction system hasa total volume of 10 μl, containing: 5 μL 2×SYBR Green Master Mix, 0.5μl forward primer (10 μM), 0.5 μl reverse primer (10 μM), 1 μl cDNA byreverse transcription, 3 μl RNase-free dH₂O. LightCycler 480fluorescence quantitative PCR instrument was used, and the PCR reactionconditions were: 95° C. for 5 min for pre-denaturation, followed by PCRamplification cycle: (1) 95° C., 10 s; (2) 55° C., 10 s; (3) 72° C., 20s; a total of 40 cycles; 40° C. for 10 s in the end to cool down. Boththe forward and reverse primers of the amplification reaction weredesigned and synthesized by Beijing Tsingke Biotechnology Co., Ltd. (U6F Primer: GCGCGTCGTGAAGCGTTC (SEQ ID NO: 23), U6 R Primer:GTGCAGGGTCCGAGGT (SEQ ID NO: 24), HJT-sRNA-m7 F Primer:TCGCGCTGAGGTAGTAGGTT (SEQ ID NO: 25), HJT-sRNA-m7 R Primer:GTGCACGCTCCGAGGT (SEQ ID NO: 26)).

3) The relative expression level was calculated by 2-ΔCt method.

3.3.3 Fluorescence In Situ Hybridization (FISH) of Small RNA

1) The medium was removed and washed 3 times with PBS (500 μl/well).

2) Fix in 4% paraformaldehyde (500 μl/well, prepared in PBS buffer) atroom temperature for 20 min.

3) Wash with 1×PBS (500 μl/well) and soak for 5 min in fresh 1×PBS (500μl/well).

4) PBS was removed, and cells were permeabilized with PK (proteinase K)buffer at room temperature for 10 min.

5) Wash with 1×PBS (500 μl/well), and fix in 4% paraformaldehyde (500μl/well, prepared in PBS buffer) at room temperature for 10 min.

6) Wash with 1×PBS, and soak for 5 min in fresh 1×PBS (500 μl/well).

7) Cells were treated with 0.1 M TEA at room temperature for 10 min.

8) Wash with 1×PBS (500 μl/well) and soak for 5 min in fresh 1×PBS (500μl/well).

9) The culture plate was placed in a hybridization cassette inhybridization buffer (50% formamide, 5×SSC, 5× Denharts, 250 μg/mL yeastRNA, 500 μg/mL herring sperm DNA) and pre-incubated at room temperaturefor 1 hour.

10) Add RNA probes (HJT-sRNA-m7 probe:5′-GCTTACAACCACACAACCTACTACCTCA-3′ (SEQ ID NO: 27), Scrambled probe:5′-CAGTACTTTTGTGTAGTACAA-3′ (SEQ ID NO: 28), U6 probe:5′-TTTGCGTGTCATCCTTGCG-3′ (SEQ ID NO: 29)) to the hybridization buffer(the concentration of RNA probes was 0.1-0.2 ng/μ1), denature at 85° C.for 5 min, and quickly place it on ice.

11) Remove the pre-hybridization buffer from step 9, and replace it withthe hybridization buffer containing the RNA probe from step 10, thenplace the plate in the hybridization cassette, and incubate overnight(12-16 hours) at 65° C.

12) Pre-heat the 0.2×SSC solution to 65° C. and wash three times with0.2×SSC (1 mL/well) for 20 min each time.

13) Add 0.2×SSC solution (1 mL/well) at room temperature and stand for 5min.

14) Aspirate 0.2×SSC, add Buffer B1 (0.1 M Tris-HCl (pH 7.4-7.5), 150 mMNaCl), and wash twice at room temperature, 5 min each time.

15) Wash three times with PBS, 5 min each time. 16) Observe underconfocal microscopy.

3.4 Effects of Traditional Chinese Medicine Extracts on Absorption andEntry of Nucleic Acids into Cells

1) 30 lipids shown in Table 1 were selected for the experiments, andexperimental groups were numbered according to the lipid numbers shownin Table 1. Lipid nucleic acid mixtures were prepared according to thereverse evaporation method and the boiling method described in Step 2.The in vitro delivery experiment was carried out using the lipid nucleicacid mixtures according to steps 3.1-3.3, and the abundance ofintracellular RNA was determined.

The experimental results were shown in FIGS. 1-4. FIGS. 1-2 indicatedthat the lipid nucleic acid mixtures prepared by the reverse evaporationmethod could successfully deliver nucleic acids to NCI-N87 and MRC-5cells; FIGS. 3-4 showed that the lipid nucleic acid mixtures prepared bythe boiling method could successfully deliver nucleic acids to MRC-5 andA549 cells.

2) Further, various lipids of Table 1 were combined. 200 μl lipidcombination in diethyl ether solution (having a concentration of 0.00326mg/mL for the lipid combination of No.1/2/4/9/18/19/20/21/22/23/24/25/26/27/28/29, 0.00652 mg/mL for the lipidcombination of No. 3/8/10/13, 0.000652 mg/mL for the lipid combinationof No. 15/16/17) and 3 μl lipid combination in chloroform solution(having a concentration of 5 mg/mL for lipid combination of No.1/2/4/9/18/19/20/21/22/23/24/25/26/27/28/29, 10 mg/mL for the lipidcombination of No. 3/8/10/13, 1 mg/mL for the lipid combination of No.5/16/17) were prepared. The above lipids were mixed in equal volume toobtain the mixed lipids and to prepare the lipid nucleic acid mixturesby the reverse evaporation method and the boiling method, respectively,as described below. The in vitro delivery experiment was carried outusing the lipid nucleic acid mixtures according to steps 3.1-3.3, andthe abundance of intracellular RNA was determined.

Preparation of Mixture of Lipid Combination and Nucleic Acid by ReverseEvaporation Method:

200 μl lipid combination in diethyl ether solution was added to 40 μlHJT-sRNA-m7 aqueous solution (5 μM) at a volume ratio of 5:1 between thelipid solution and the RNA, and sonicated for 3 min; diethyl ether wasremoved by evaporation at 55° C., and then 200 μl DEPC water was addedfor hydration to obtain lipid nucleic acid mixture.

Manufacture of Mixture of Lipid Combination and Nucleic Acid by BoilingMethod:

3 μl lipid combination in chloroform solution was mixed with 100 μlHJT-sRNA-m7 aqueous solution (2 μM), and heated at 100° C. for 30 min.

The experimental results were shown in FIGS. 5-8. FIGS. 5-6 demonstratedthe mixture of lipid combination and nucleic acid prepared by thereverse evaporation method could successfully facilitate nucleic acidentry into a target cell; FIGS. 7-8 demonstrated the mixture of lipidcombination and nucleic acid by the boiling method could successfullyfacilitate nucleic acid entry into a target cell.

3) The different types of lipids shown in Table 1, e.g., TG mixture, DGmixture and the like, were combined, and used for the preparation oflipid nucleic acid mixture by the reverse evaporation method and theboiling method, respectively. The in vitro delivery experiment wascarried out using the lipid nucleic acid mixtures according to steps3.1-3.3, and the abundance, intracellular localization and targetedregions of intracellular RNA were determined.

Different types of lipids were combined as follows:

Combination 1: combination of lipids No. 1-32, No.1/2/3/4/6/8/9/10/13-32 without lipids No. 5, 7, 11, and 12;

Combination 2: combination 1 without lipid No. 29; Combination 3:combination 1 without lipids No. 1, 2, 3, 19;

Combination 4: combination 1 without lipids No. 4, 14;

Combination 5: combination 1 without lipids No. 6, 9, 10, 13, 15, 16,18, 20-28, 32;

Combination 6: combination 1 without lipid No. 8; Combination 7:combination 1 without lipids No. 17, 30, 31;

FA: lipid No. 29;

DG combination: combination of lipids No. 1, 2, 3, 19;

Cer combination: combination of lipids No. 4, 14;

TG combination: combination of lipids No. 6, 9, 10, 13, 15, 16, 18,20-28, 32;

PE combination: lipid No. 8;

So combination: combination of lipids No. 17, 30, 31.

The experimental results were shown in FIGS. 9-10. The results showedthat different types of lipid combination (e.g., mixture of TG, mixtureof DG, etc.) by different methods (boiling or reverse evaporation) couldpromote nucleic acid entry into a target cell.

4) Further, lipids No. 11 and No. 12 were selected for experiments toinvestigate the efficiency of the lipids to deliver nucleic acidfragments having different sequences, as well as the localization andthe targeted gene regions of the nucleic acids. The protocols were asfollows:

Mixtures of soybean PC, lipid No. 11 (18:0/18:2) and lipid No. 12(16:0/18:2), and various small RNA (see below Table 3) were prepared bythe reverse evaporation method and then added into A549 cell line (thefinal concentration of sRNA was 200 nM); the negative control group(control) was directly added with the same concentration of sRNA; thepositive control group (RNAimax) was transfected with LipofectamineRNAimax (transfection reagent 6 μl/well). The abundance of sRNA in thecells was detected by Taqman probe after 3 hours, and the relativeexpression level of sRNA was calculated by 2-ΔCt method.

The experimental results were shown in FIGS. 11-13. FIGS. 11A-C showedthat compared with the control, the two lipids (lipid No. 11 (18:0/18:2)and lipid No. 12 (16:0/18:2)) could effectively facilitate nucleic acidmolecules of different sequences entry into various cells; FIG. 12showed that nucleic acids that were delivered by lipid No. 11(18:0/18:2)and lipid No. 12 (16:0/18:2) entered into cytoplasm and were primarilylocalized in cytoplasm. In addition, with reference to FIG. 13, theinventor unexpectedly found that both lipids No. 11 and No. 12 promotedthe entry of small fragments of nucleic acids, which acted on thewild-type 3′UTRs of their target genes and reduced the relativeexpression level of the Luciferase with the wild-type 3′UTR in thetarget gene, while did not act on the mutated 3′UTR of their targetgenes. It can be used as a means for the delivery of nucleic acid drug.

4. In Vivo Delivery Experiments of Lipid Nucleic Acid Mixture

4.1 Experimental Steps

1. Preparation of lipid nucleic acid mixture: the mixture of lipid No.11 or No. 12 with nucleic acid, and the mixture of the lipid combinationof No. 1/2/4/9/14/18/19/20/21/22/23/24/25/26/27/28/29/30/32, No.3/8/10/11/12/13, and No. 6/15/16/17/31 with nucleic acid were preparedby reverse evaporation and boiling method (refer to steps 2.1-2.2).

2. Gavage were performed on 6-8 weeks old male C57 mice: 200 μl/aminal,grouped as follows:

(1) Control group (free uptake group): no treatment or HJT-sRNA-m7 wasgiven by gavage;

(2) Lipid No. 11 (18:0/18:2) group: lipid No. 11 (18:0/18:2) or amixture of lipid No. 11 (18:0/18:2) and HJT-sRNA-m7 were given bygavage;

(3) Lipid No. 12 (16:0/18:2) group: lipid No. 12 (16:0/18:2) or amixture of lipid No. 12 (16:0/18:2) and HJT-sRNA-m7 were given bygavage;

3. Sample collection: 6 hours after gavage, mouse whole blood (500 μl)and lung (110 mg) were collected by 1.5 mL TRIzol-LS or 3 mL TRIzol,respectively, homogenized and then frozen under −80° C. for storage.

4. Total RNA extraction: (1) Add TRIzol or TRIzol-LS lysis buffer (SigmaCorporation) to the cells, which were then left at room temperature for5 min to be fully lysed (for mouse lung tissue, to 100 mg tissue wasadded 1.0 mL TRIzol lysis buffer, and the solution was ground with ahomogenizer, centrifuged at 12,000 rpm, 4° C. for 10 min to remove thetissue precipitate which was not homogenized; for the mouse whole blood,to 500 μl of whole blood was added 1.5 mL TRIzol-LS lysis buffercentrifuged at 12,000 rpm, 4° C. for 10 min to remove the precipitatethat has not been fully cleaved); (2) 12,000 rpm, 4° C., centrifuge for5 min, and discard the precipitate; (3) add chloroform at a ratio of 200μl/mL TRIzol, vortex to mix, allow to stand at room temperature for 15min. (4) 12,000 rpm, 4° C., centrifuge for 15 min, pipette the upperaqueous phase to another centrifuge tube; (5) Repeat step 4, add equalamount of chloroform into the upper aqueous phase, mix well, and allowto stay for 10 min at room temperature, 12,000 rpm, 4° C., centrifugefor 15 min; (6) Draw the upper aqueous phase to a fresh EP tube, addisopropanol at a ratio of 0.5 ml/mL TRIzol, mix, and allow to stay atroom temperature for 5-10 min; (7) 12,000 rpm, 4° C., centrifuge for 10min, discard the supernatant; (8) add 1 mL 75% ethanol, gently shake thecentrifuge tube, suspend the precipitate; (9) 8000 g, 4° C., centrifugefor 5 min, discard the supernatant as much as possible; and (10) dry atroom temperature for 5-10 min and dissolve the RNA sample with 50 μlDEPC-treated H₂O.

5. RT-qPCR detection: see the method described in above Sections 3.3.1and 3.3.2.

4.2 Experimental Results

With reference to FIG. 14, the inventor unexpectedly discovered thatlipid No. 11 (18:0/18:2) and lipid No. 12 (16:0/18:2) could promoteentry of small fragments of nucleic acids into the blood and lung by(non-invasive) gavage, which can be used as a means for the delivery ofnucleic acid drug. Surprisingly, the lipid nucleic acid mixture obtainedby direct boiling method achieved a significant delivery effect.

With reference to FIG. 15, the inventor surprisingly found that, themixture of 28 lipids could facilitate entry of small fragments ofnucleic acids into the blood by the (non-invasive) gavage, which can beused as a means for the delivery of nucleic acid drug. Surprisingly, themixture of lipid combination with nucleic acid obtained by directboiling method achieved a significant delivery effect.

Examples for Lipids Shown in Table 1 as No. 1-71

Method

1. Extraction of Lipids from Traditional Chinese Medicine

1.1 Decoction of Chinese Medicine

1) 100 g Chinese medicine decoction pieces (Rhodiola crenulata,Taraxacum mongolicum, Lonicera japonica and Andrographis paniculata,purchased from Beijing Tongrentang pharmacy) was added to 1000 mL ddH₂Oand soaked for 30 min.

2) The Chinese medicine decoction pot was boiled for 15 min with intenseheating, and for 20 min with slow heating.

3) 400 mL of the heated Chinese medicine soup was added to a rotaryevaporator, and was concentrated to 100 mL at 60° C., 60 rpm, 30 min.

1.2 Lipid Extraction

1) To the 160 mL Chinese medicine soup (concentrated by rotaryevaporator) was added chloroform-methanol mixture(chloroform:methanol=1:2, v/v) 600 mL to makechloroform:methanol:water=1:2:0.8, and stirred for 10-15 min to mix.

2) 200 mL chloroform was add to the Erlenmeyer flask and stirred for 10min to mix.

3) 200 ml ddH₂O was added to the Erlenmeyer flask to makechloroform:methanol:water=2:2:1.8, stirred for 10 min to mix.

4) The liquid of upper layer and the insoluble substances ofintermediate layer was removed, and the chloroform layer of lower layerwas taken out and stored at −40° C.

1.3 HPLC-MS/MS Identification of Lipid Components

Instrument Setup

1) Chromatographic Setup:

Instrument: Ultimate 3000; column: Kinetex C18 (100×2.1 mm, 1.9 μm);column temperature: 45° C.; mobile phase A: acetonitrile:water (v/v,60:40), the solution containing 10 mmol/L ammonium formate, mobile phaseB: acetonitrile:isopropanol (10:90, v/v), the solution containing 10mmol/L ammonium formate and 0.1% formic acid. Flow rate: 0.4 mL/min;injection volume: 4 μl.

2) Mass Spectrometry Parameters:

a) Positive mode: Heater Temp 300° C., Sheath Gas Flow rate, 45 arb, AuxGas Flow Rate, 15 arb, Sweep Gas Flow Rate, 1 arb, spray volt age, 3.0KV, Capillary Temp, 350° C., S-Lens RF Level, 30%. Scan ranges:200-1500.

b) Negative mode: Heater Temp 300° C., Sheath Gas Flow rate, 45 arb, AuxGas Flow Rate, 15 arb, Sweep Gas Flow Rate, 1 arb, spray voltage, 2.5KV, Capillary Temp, 350° C., S-Lens RF Level, 60%. Scan ranges:200-1500.

1.4 Identification of the Lipids Derived from Chinese Medicine

The lipid components were identified by HPLC-MS/MS, and a total of 138lipid components derived from traditional Chinese medicine wereidentified, among which 125 were identified in positive mode and 13 innegative mode. The following experiments was performed on the compounds1-69 shown in Table 1. It should be noted that the lipids tested belowwere all commercially purchased or commercially synthesized, and used asdescribed in Table 1-1.

2. Manufacture of Lipid Nucleic Acid Mixture

2.1 Reverse Evaporation Method:

100 μl lipid in diethyl ether solution was prepared, and groupedaccording to the lipid numbers shown in Table 1 (the lipidconcentrations are shown in the table below). To the lipid solution wasadded 20 μl nucleic acid solution (HJT sRNA or siRNA) at the volumeratio of 5:1, and sonicated for after 3 min. The diethyl ether wasremoved by evaporation at 55° C., and then 100 μl DEPC water was addedfor hydration to give nucleic acid lipid mixture.

TABLE 3 Single lipid or Concentration/ Figure lipid combination (mg/mL) 51 8 + 12 = 1:2 No. 8 0.0833 No. 12 0.1667  71 38 + 12 + 37 = 4:1:1 No.38 0.2 No. 12 0.05 No. 37 0.05 76/77/79/ No. 41 No. 41 0.25  82  87 40 +12 + 41 = 2:4:3 No. 40 0.0667 No. 12 0.1333 No. 41 0.1 88 left 12 + 41 =1:6 No. 12 0.0428 No. 41 0.2571 88 left 12 + 41 = 1:1 No. 12 0.15 No. 410.15 89 left 12 + 41 = 6:1 No. 12 0.2571 No. 41 0.0428 89 right 4 + 12 +41 = 1:1:1 No. 4 0.1 No. 12 0.1 No. 41 0.1  90 4 + 12 + 41 = 1:1:1 No. 40.1 No. 12 0.1 No. 41 0.1  93 No. 38 No. 38 0.25 99/100/102 No. 40 No.40 0.25 104/105 No. 39 No. 39 0.25 106 No. 60 No. 60 0.25 107 No. 62 No.62 0.25

2.2 Boiling Method:

100 μL of the nucleic acid solution (HJT sRNA or siRNA) was added to 2-5μL of the lipid solution (the concentration was shown in Table 1),mixed, and heated at 80-100° C. for 15-30 min to give nucleic acid lipidmixture.

3. In Vitro Delivery Experiment of Lipid Nucleic Acid Mixture

3.1 Real-Time Quantitative PCR (RT-qPCR) Detection of IntracellularExpression of Nucleic Acids Delivered by Lipid.

3.1.1 MRC-5 cell (pulmonary embryonic fibroblast), A549 cell (human lungadenocarcinoma cell), Caco-2 cell (human colon adenocarcinoma cell)(purchased from the Cell Resource Center of the Institute of BasicMedical Sciences, Chinese Academy of Medical Sciences) were cultured tologarithmic growth phase, then plated into 12-well plates at a celldensity of 6×10⁵/1 mL medium/well; MRC-5 and Caco-2 cells were culturedin Eagle's MEM medium (MEM, Gibco); A549 cells were cultured in Ham'sF-12 medium (HyClone); followed by incubation overnight at 37° C., andthe follow-up experiments were performed after the cells were attachedto the walls.

3.1.2 Experimental groups were as follows:

1) naive group: it referred to untreated cells, and this group served asa blank control group.

2) RNAimax treatment group: 2 μl Lipofectamine™RNAimax transfectionreagent (full name of Lipofectamine RNAiMAX, Invitrogen, Thermo FisherScientific) and HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium (purchased from Invitrogen, Thermo Fisher Scientific)respectively and then the two were mixed, allowed to stand for 15 min,added into cells and then mixed. The final concentration of HJT-sRNA-m7was 100 nM; this group served as a positive control group.

3) Free uptake group: HJT-sRNA-m7 solution was directly added (the finalconcentration was 100 nM), and the group served as a negative controlgroup.

4) Lipid nucleic acid mixture: the mixture of lipid and HJT-sRNA-m7prepared from the step 2 were added into cells and mixed, and the finalconcentration of HJT-sRNA-m7 was 100 nM.

3.1.3 After co-incubation with cells for 12-24 hours, the cells werewashed twice with PBS. The cells were harvested with TRIzol lysis buffer(purchased from Sigma-Aldrich), and total RNA was extracted. Theabundance of HJT-sRNA-m7 that entered the cells was detected by RT-qPCR;the protocols were as follows:

1) Extraction of total cellular RNA:

A. To the cells cultured in a 12-well plate (about 1×10⁶ cells/well) wasadded 1 mL TRIzol lysis buffer in each well, and then placed on ice.After to all the samples was added TRIzol, they were allowed to stand atroom temperature for 5 min to allow them fully lysed.

B. Centrifuge at 4° C., 12,000 rpm for 5 min, discard the pellet andtransfer TRIzol to a fresh centrifuge tube;

C. Add chloroform at a ratio of 200 μL chloroform/mL TRIzol, shake well,mix and allow to stand for 5 min at room temperature;

D. Centrifuge at 4° C., 12,000 rpm for 15 min;

E. Pipette the upper aqueous phase into another centrifuge tube, addisopropanol at a ratio of 0.5 mL isopropanol/mL TRIzol and allow tostand at room temperature for 5-10 min;

F. Centrifuge at 4° C., 12,000 rpm for 15 min, discard the supernatant,and allow the RNA to precipitate to the bottom of the tube;

G. Add 1 mL 75% ethanol, gently shake the tube to suspend theprecipitate;

H. Centrifuge at 4° C., 12,000 rpm for 10 min, discard the supernatant,add 1 mL 75% ethanol, gently shake the centrifuge tube to suspend theprecipitate;

I. Centrifuge at 4° C., 12,000 rpm for 10 min, discard the supernatant,dry at room temperature, dissolve the RNA sample with 50 μL RNase-freeH₂O, and quantify the RNA concentration by the measurement of OD value.

2) Total RNA was reverse transcribed to cDNA: Reverse Transcription Kit(High-Capacity cDNA Reverse Transcription Kits, Applied Biosystems, cat.no. 4368813) was used to reverse transcribe sRNA to cDNA by stem-loopmethod (see, e.g. Real-time quantification of microRNAs by stem-loopRT-PCR, Nucleic Acids Res. 2005 Nov. 27; 33(20):e179, incorporated byreference herein). The reverse transcription system was as follows:template RNA (150 ng/μL) 10 μL, 10×RT buffer 2.0 μL, 25×dNTP Mix (100mM) 0.8 μL, U6 RT stem-loop primer 2.0 μL, HJT-sRNA-m7 RT stem-Loopprimer 2.0 μL, MultiScribe™ reverse transcriptase 1.0 μL, RNaseinhibitor 1.0 μL, nuclease-free H₂O 1.2 μL, loaded into a PCR reactorafter brief centrifugation. The reaction conditions were as follows: (1)25° C., 10 min; (2) 37° C., 120 min; (3) 85° C., 5 min; (4) 4° C.,termination of reaction. 20 μl RNase-free ddH₂O was added to make up thefinal volume to 40 μl after the reaction. The stem-loop primer used inthe reverse transcription process was synthesized by Beijing TsingkeBiotechnology Co., Ltd. (U6 RT primer, because the quantification ofsmall RNA by RT-qPCR reaction can only be relative, so U6 was used as astandard reference gene for calculating relative expression level):GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAAAT ATG (SEQ ID NO: 21);HJT-sRNA-m7 RT stem-loop primer:GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACGCTTAC AA (SEQ ID NO: 22)).

3) Quantitative PCR amplification reaction: the qPCR reaction system hada total volume of 10 μl, containing: 5 μL 2×SYBR Green Master Mix, 0.5μl forward primer (10 μM), 0.5 μl reverse primer (10 μM), 1 μl cDNA byreverse transcription, 3 μl RNase-free dH₂O. LightCycler 480fluorescence quantitative PCR instrument was used, and the PCR reactionconditions were: 95° C., pre-denaturation for 5 min, followed by PCRamplification cycle: (1) 95° C., 10 s; (2) 55° C., 10 s; (3) 72° C., 20s; a total of 40 cycles; 40° C. for 10 s in the end to cool down. Boththe forward and reverse primers of the amplification reaction weredesigned and synthesized by Beijing Tsingke Biotechnology Co., Ltd. (U6forward primer: GCGCGTCGTGAAGCGTTC (SEQ ID NO: 23), U6 reverse primer:GTGCAGGGTCCGAGGT (SEQ ID NO: 24), HJT-sRNA-m7 forward primer:TCGCGCTGAGGTAGTAGGTT (SEQ ID NO: 25), HJT-sRNA-m7 reverse primer:GTGCACGCTCCGAGGT (SEQ ID NO: 26)).

4) 2-ΔCt method (relative gene expression level=2-(Ct target gene-Ctinternal reference gene)) was used to calculate the relative amount ofentry (single or double stranded RNA).

3.2 Real-Time Quantitative PCR (RT-qPCR) Detection of mRNA ExpressionLevels

3.2.1 THP-1 cell (human monocyte) was cultured to logarithmic growthphase, then plated into 12-well plates at a cell density of 6×10⁵/1 mLmedium/well; THP-1 cells were cultured in RPMI-1640 medium (HyClone);the cells were incubated overnight at 37° C., and the follow-upexperiments were performed after the cells were attached to the walls.

3.2.2 Experimental groups were as follows:

1) naive group: referred to untreated THP-1 cells, and this group servedas a blank control group.

2) RNAiMAX treatment group: 2 μl Lipofectamine™RNAimax transfectionreagent (Invitrogen, Thermo Fisher Scientific) and nucleic acid solution(TNFα siRNA) were diluted in 100 μl opti-MEM medium (Invitrogen, ThermoFisher Scientific) respectively and then the two were mixed, allowed tostand for 15 min, added into cells, and then mixed. The finalconcentration of nucleic acid was 400 nM; this group served as apositive control group.

3) Free uptake group: nucleic acid solution (TNFα siRNA) was directlyadded (the final concentration was 400 nM), the group served as anegative control group.

4) Lipid nucleic acid mixture: the mixture of lipid and nucleic acidprepared from the step 2 were added into cells and mixed, and the finalconcentration of nucleic acid was to 400 nM.

3.2.3 After 24 hours of treatment, the cells were stimulated with 1μg/mL E. coli LPS (Lipopolysaccharide, LPS, Escherichia coli 0111:B4,L4391, Sigma-Aldrich), and harvested using TRIzol lysis buffer after 9hours to extract total RNA. The mRNA expression level of TNF-α (thetarget genes of the subsequent examples varied case by case and wereindicated in the Figures) was determined by RT-qPCR (SYBR Green dyemethod), and the protocols were as follows:

1) Extraction of the total RNA from cells: the procedures were the sameas the method of extracting total RNA in Section 3.1.3.

2) Total RNA was reverse transcribed to cDNA: Reverse Transcription Kit(High-Capacity cDNA Reverse Transcription Kits, Applied Biosystems, cat.no. 4368813) was used to reverse transcribe the total RNA to cDNA. Thereverse transcription system was as follows: template RNA (150 ng/μL) 10μL, 10×RT buffer 2.0 μL, 25×dNTP Mix (100 mM) 0.8 μL, random primers 2.0μL, MultiScribe™ reverse transcriptase 1.0 RNase inhibitor 1.0 μL,nuclease-free H₂O 3.2 μL, loaded into a PCR reactor after briefcentrifugation. The reaction conditions were as follows: (1) 25° C., 10min; (2) 37° C., 120 min; (3) 85° C., 5 min; (4) 4° C., termination ofreaction. 20 μl RNase-free dd H₂O was added to make up the final volumeto 40 μl after the reaction.

3) Quantitative PCR amplification reaction: the total volume of qPCRreaction system was 10 μl, containing: 5 μL 2×SYBR Green Master Mix, 0.5μl forward primer (10 μM), 0.5 μl reverse primer (10 μM), 1 μl cDNA byreverse transcription, 3 μl RNase-free dH₂O. LightCycler 480fluorescence quantitative PCR instrument was used, the PCR reactionconditions were: 95° C., pre-denaturation for 5 min, followed by PCRamplification cycle: (1) 95° C., 10 s; (2) 55° C., 10 s; (3) 72° C., 20s; a total of 40 cycles; 40° C. for 10 s in the end to cool down. Boththe forward and reverse primers of the amplification reaction weredesigned and synthesized by Beijing Qingke Biotechnology Co., Ltd. Theprimer sequences were as follows: forward primer for internal referencegene UBC: CTGGAAGATGGTCGTACCCTG (SEQ ID NO: 30), reverse primer forinternal reference gene UBC: GGTCTTGCCAGTGAGTGTCT (SEQ ID NO: 31);forward primer for target gene TNF-α: CTGCCCCAATCCCTTTATT (SEQ ID NO:32): reverse primer for target gene TNF-α: CCCAATTCTCTTTTTGAGCC (SEQ IDNO: 33).

4) The relative expression level was calculated 2-ΔCt method asdescribed above.

3.3 Western Blot Detection of Protein Expression Levels

3.3.1 MRC-5 cell (pulmonary embryonic fibroblast), and A549 cell (humanlung adenocarcinoma cell) were cultured to logarithmic growth phase, andthen plated into 12-well plates at a cell density of 6×10⁵/1 mLmedium/well; MRC-5 cells were cultured in Eagle's MEM medium (MEM,Gibco); A549 cells were cultured in Ham's F-12 medium (HyClone);followed by incubation overnight at 37° C., and the follow-upexperiments were performed after the cells were attached to the walls.

3.3.2 Experimental groups were as follows:

1) Naive group: it referred to the untreated cells, and this groupserved as a blank control group.

2) RNAiMAX treatment group: 2 μl Lipofectamine™RNAimax transfectionreagent (Invitrogen, Thermo Fisher Scientific) and nucleic acid solutionwere diluted in 100 μl opti-MEM medium (Invitrogen, Thermo FisherScientific) respectively and then the two were mixed, allowed to standfor 15 min, added into cells, and then mixed. The final concentration ofnucleic acid was 400 nM; this group served as a positive control group.

3) Free uptake group: the nucleic acid solution was directly added (thefinal concentration was 400 nM), and the group served as a negativecontrol group.

4) Lipid nucleic acid mixture: the mixture of lipid and nucleic acidprepared from the step 2 were added into cells and mixed, and the finalconcentration of nucleic acid was 400 nM.

3.3.3 After 24 hours of treatment, the cells were stimulated with thestimulant (1 μg/mL poly (I:C) (P1530, Sigma-Aldrich) as double-strandedRNA viruses mimetics) or 3 ng/mL transforming growth factor TGFβ1 (PeproTech)). The cells were harvested using strong RIPA lysis buffer, andafter incubation for some time, Western blot was used to detect theprotein expression level of the related genes (the types of the relatedgene varied case by case and were indicated in the correspondingFigures) (the protein expression level of REL-A was detected 24 hoursafter the A549 cells were stimulated by poly(I:C) with β-actin as theinternal reference protein; the protein expression levels of fibronectinand α-SMA were detected 72 hours after MRC-5 cells were stimulated withTGF-β1 with GAPDH as the internal reference protein; the proteinexpression of the corresponding knockdown genes was detected in thesiRNA delivery assay with β-actin as the internal reference protein).The protocols were as follows:

1) Collection of protein samples and determination of the concentrationby BCA method.

A. Discard the medium, add 1 mL PBS buffer into each well of the 12-wellplate to wash the cells once, add 100 μL precooled strong RIPA lysisbuffer into each cell, scrap off the cells with a pipette tip andtransfer to a centrifuge tube, place and keep on ice for 20 min forlysis;

B. Centrifuge at 4° C., 12,000 rpm for 10 min, transfer the supernatantto a frech centrifuge tube;

C. Mix BCA reagent A and B (50:1, v/v) thoroughly to prepare a BCAworking solution;

D. Add 25 μL of the freshly prepared BSA standard solution and thesamples to be tested to a 96-well plate, add 200 μL BCA working solutionto each well and mix well; incubate at 37° C. for 30 min;

E. Measure the absorbance at 562 nm using an ultravioletspectrophotometer (Synergy 4 multi-function microplate reader), andcalculate the protein concentration in the samples according to thestandard curve;

F. Adjust the concentration of the samples with RIPA lysis buffer andloading buffer so that the concentration of each sample was the same;

G. Denaturation at 95° C. for 10 min.

2) Western blot

A. Gel preparation: a resolving gel (lower layer gel) with aconcentration of 10% and stacking gel (upper layer gel) with aconcentration of 5% were used. The lanes were made with a 15-well comb,and equal amounts of protein were loaded in each lane;

B. Protein electrophoresis: add electrophoresis buffer and use aninitial voltage of 80V for electrophoresis; when the bromophenol bluedye reach the resolving gel, increase the voltage to 120V and continueelectrophoresis until the bromophenol blue dye reach the bottom orcompletely out of the resolving gel;

C. Wet transfer: make the assembly in the following order: transfer pad(anode)-sponge-filter paper-gel-PVDF membrane-filterpaper-sponge-transfer pad (cathode); install the assembly and put thewhole transfer device at 4° C. cold chamber; set constant current at 300mA for a 120 min transfer;

D. Blocking: place the membrane in a 3% BSA blocking solution after thetransfer and block at room temperature for 1 hour;

E. Primary antibody incubation: transfer the blocked PVDF membrane tothe hybridization bag, add 3% BSA blocking solution containing thecorresponding primary antibody (the primary antibody informations wereas follows), remove the bubbles in the bag, and incubate overnight at 4°C.

TABLE 4 Primary Dilution ratio of Secondary Antibody Company Cat. No.primary antibody Antibody fibronectin Sigma Aldrich F7387 1:2000 M α-SMAAbcam ab7817 1:1000 M GAPDH Protein Tech 60004-1-1g 1:5000 M LAMP1 SantaCruze sc-20011 1:1000 M LAMP2 Santa Cruze sc-18822 1:1000 M XRN2 SantaCruze sc-365258 1:2000 M CPSF4 Protein Tech 15023-1- 1:1000 R AP Ssu72CST 12816s 1:1000 R NF-κB CST 4764S 1:2000 R β-actin Sigma Aldrich A54411:5000 M

F. Membrane wash: take out the PVDF membrane and wash the membrane 3times with TBST for 10 min each time;

G. Secondary antibody incubation: discard TBST, add 3% BSA blockingsolution containing goat anti-rabbit or goat anti-mouse secondaryantibody with horseradish peroxidase (HRP) (purchased from HangzhouLianke Biotechnology Co., Ltd.) (dilution ratio of secondary antibodywas 1:5000), incubate for 1 hour at room temperature;

H. Membrane wash: wash the membrane 3 times with TBST for 10 min eachtime;

I. Developing: prepare Western developing solution (1:1, V/V, MerckMillipore, ECL chemiluminescence developing solution purchased fromMillipore), and add the prepared developing solution evenly to the sidethe membrane that is bound to the proteins; carefully wrap the film withplastic wrap and observe after developing;

J. Analysis: analysis was performed using Image J software.

4. In Vivo Delivery Exeperiments of Lipid Nucleic Acid Mixture

4.1 Experimental Steps:

1) Preparation of lipid nucleic acid mixture: boiling method was used.To 400 μL

HJT-sRNA-m7 (5 nmol) single-stranded RNA in DEPC-treated solution wasadded 9 μL or 18 μL lipid combinations (lipid PE (No. 38) & LPC (No. 37)& TG (No. 32), 4:2:3, V/V/V) respectively, mixed and heated at 100° C.for 30 min.

2) Intragastric administration of RNA in 6-8 weeks old male C57BL/6Jwild type mice: HJT-sRNA-m7 aqueous solution or the mixture solution oflipid and HJT-sRNA-m7 were administered using a gavage needle, 400μL/animal (HJT)-sRNA-m7, 5 nmol/animal). The groups were as follows:

A. Control group (naive group): mice that did not receive any treatment;

B. Negative control group (lipid group): intragastric administration of9 μL lipid combinations (lipid PE (No. 38) & LPC (No. 37) & TG (No. 32),4:2:3, V/V/V);

C. Free uptake group: direct intragastric administration of HJT-sRNA-m7single-stranded RNA solution;

D. Lipid and nucleic acid mixture group: intragastric administration ofthe mixture of lipid combination and HJT-sRNA-m7 single-stranded RNA.

3) Sample collection: 3 hours after intragastric administration, themouse whole lung was lysed with 3 mL TRIzol, homogenized and frozen at−80° C.

4) Total RNA extraction:

A. Add 3.0 mL TRIzol lysis buffer to mouse lung tissue, grind with ahomogenizer, centrifuge at 12,000 rpm, 4° C., for 10 min, remove thetissue precipitate that failed to homogenize;

B. Add chloroform at a ratio of 200 μl/mL TRIzol, shake well to mix, andkeep at room temperature for 15 min.

C. centrifuge at 12,000 rpm, 4° C., for 15 min, pipette the upperaqueous phase to another centrifuge tube;

D. Repeat the above step, add equal amount of chloroform to the upperaqueous phase, mix well, and keep for 10 min at room temperature;

E. 12,000 rpm, 4° C., centrifuge for 15 min;

F. Draw the upper aqueous phase to a fresh EP tube, add isopropanol aratio of 0.5 ml/mL TRIzol, mix and keep at room temperature for 5-10min;

G. 12,000 rpm, 4° C., centrifuge for 15 min, discard the supernatant;

H. Add 1 mL 75% ethanol, gently shake the centrifuge tube, and suspendthe precipitate;

I. 12,000 rpm, 4° C., centrifuge for 10 min, discard the supernatant asmuch as possible;

J. Dry at room temperature for 5-10 min and dissolve the RNA sample with50 μl DEPC-treated H₂O.

5) Detection of the abundance of HJT-sRNA-m7 by RT-qPCR (SYBR Greenuniversal dye method).

Unless otherwise indicated, the single stranded HJT-sRNA-m7 solutionrefers to single-stranded HJT-sRNA-m7 in DEPC-treated aqueous solution.The double-stranded HJT-sRNA-m7 solution refers to a double-strandedHJT-sRNA-m7 in DEPC-treated aqueous solution.

Example 1-1: Delivery of Single-Stranded Nucleic Acids into MRC-5 Cellby Different Types of Lipid Combination

1. Experimental Groups:

1) Naive group: untreated MRC-5 cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 in DEPC-treated aqueous solution werediluted in 100 μl opti-MEM medium, respectively, and then the two weremixed, allowed to stand for 15 min, added into cells, and then mixed.The final concentration of single-stranded HJT-sRNA-m7 was 200 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 200 nM);

4) Lipid nucleic acid mixture: mixtures of 3 μL single lipid or lipidcombination and HJT-sRNA-m7 single-stranded nucleic acid solutiontreated by boiling method were added to the cells and mixed. The finalconcentration of RNA was 200 nM.

2. Experimental Procedures

1) Boiling method conditions: to 100 μL single-stranded HJT-sRNA-m7solution was added 3 μL single lipid or lipid combination in chloroformsolution (lipid No. 1/2/4/9/14/18/19/20/21/22/23/24/25/26/27/28/29/30/32in chloroform solution having a concentration of 5 mg/mL, lipid No.3/8/10/11/12/13/33/34/35/36 in chloroform solution having aconcentration of 10 mg/mL, lipid No. 6/15/16/17/31 in chloroformsolution having a concentration of 1 mg/mL), and heated at 100° C. for30 min;

a) Lipid combination:

b) MG (monoglyceride): 3 μL lipid No. 34;

c) DG (diglyceride): 3 μL mixture of equal volume of lipids No.1/2/3/19/35 in chloroform solution;

d) TG (triglyceride): 3 μL mixture of equal volume of lipids No.6/9/10/13/15/16/18/20/21/22/23/24/25/26/27/28/32/33 in chloroformsolution;

e) LPC (Lysophosphatidylcholine): 3 μL mixture of equal volume of lipidsNo. 36/37 in chloroform solution;

f) PC (phosphatidylcholine): 3 μL mixture of equal volume of lipids No.11/12 in chloroform solution;

g) PE (phosphatidylethanolamine): 3 μL mixture of equal volume of lipidsNo. 8/38 in chloroform solution;

h) Cer (Ceramides): 3 μL mixture of equal volume of lipids No. 4/14 inchloroform solution;

i) So (Sphingoshine): 3 μL mixture of equal volume of lipids No.17/30/31 in chloroform solution;

j) FA (fatty acid): 3 μL lipid No. 29;

k) Mixture: 3 μL mixture of equal volume of lipids No. 1-36 (without No.5/7) in chloroform solution;

l) Mixture 1: 3 μL mixture of equal volume of lipids No. 1-36 (withoutNo. 5/7/34) in chloroform solution;

m) Mixture 2: 3 μL mixture of equal volume of lipids No. 1-36 (withoutNo. 5/7/1/2/3/19/35) in chloroform solution;

n) Mixture 3: 3 μL mixture of equal volume of lipids No. 1-36 (withoutNo. 5/7/6/9/10/13/15/16/18/20/21/22/23/24/25/26/27/28/32/33) inchloroform solution;

o) Mixture 4: 3 μL mixture of equal volume of lipids No. 1-36 (withoutNo. 5/7/36/37) in chloroform solution;

p) Mixture 5: 3 μL mixture of equal volume of lipids No. 1-36 (withoutNo. 5/7/11/12) in chloroform solution;

q) Mixture 6: 3 μL mixture of equal volume of lipids No. 1-36 (withoutNo. 5/7/8) in chloroform solution;

r) Mixture 7: 3 μL mixture of equal volume of lipids No. 1-36 (withoutNo. 5/7/4/14) in chloroform solution;

s) Mixture 8: 3 μL mixture of equal volume of lipids No. 1-36 (withoutNo. 5/7/29) in chloroform solution;

2) Experimental conditions: the final concentration of HJT-sRNA-m7 was200 nM. 12 hours after being added to the cells, the amount ofHJT-sRNA-m7 that entered the cells was detected by RT-qPCR method (SYBRGreen Universal dye method). For the protocols, see “Real-timequantitative PCR detection of intracellular expression of nucleic acidsdelivered by lipid”. The experiments were all performed in triplicates.

Conclusions: The results showed that the above lipid combinations wereall effective in delivering nucleic acids into cells as compared to thefree uptake group (see FIG. 16), having the potential of improving theefficiency of the delivery of nucleic acid drug in clinical settings.Nucleic acids that were mediated by the mixture 2, mixture 3, mixture 5,mixture 7 entered into MRC-5 cells in higher amounts.

Example 1-2: Delivery of Single-Stranded Nucleic Acids into MRC-5 Celland Caco-2 Cell by Lipid Combination

1. Experimental Groups:

Cells to be tested were MRC-5 cell and Caco-2 cell.

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and the two were mixed, allowed to stand for 15min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 200 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 200 nM);

4) Treatment group with single lipid and nucleic acid: a mixture of 3 μLsingle lipid (No. 1 or 8 or 12) and the HJT-sRNA-m7 single-strandednucleic acid solution that was treated by boiling method was added tothe cells and mixed, and the final concentration of RNA was 200 nM;

5) Treatment group with lipid combination mixture and nucleic acidmixture: a mixture of 3 μL lipid combination (No. 1/8/12 mixed in equalvolumes) and HJT-sRNA-m7 single-stranded nucleic acid solution treatedby boiling method was added to the cells and mixed, and the finalconcentration of RNA was 200 nM;

6) Treatment group with lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (a mixture of 2 μL single lipid No. 1or No. 8 or No. 12 and 1 μL of the following types of lipids (MG, DG,TG, LPC, Cer, So, or FA)) and HJT-sRNA-m7 single-stranded nucleic acidsolution that were treated by boiling method was added to the cells andmixed, and the final concentration of RNA was 200 nM. In FIGS. 17A and17B, the treatment groups were collectively represented as No. 1 2μL+mix 1 μL, No. 8 2 μL+mix 1 μL, and No. 12 2 μL+mix 1 μL, wherein,within the horizontal line, MG represented 2 μL single lipid of No. 1 orNo. 8 or No. 12+1 μL MG, DG represented 2 μL single lipid of No. 1 orNo. 8 or No. 12+1 μL DG, TG represented 2 μL single lipid of No. 1 orNo. 8 or No. 12+1 μL TG, LPC represented 2 μL single lipid of No. 1 orNo. 8 or No. 12+1 μL LPC, Cer represented 2 μL single lipid of No. 1 orNo. 8 or No. 12+1 μL Cer, So represented 2 μL single lipid of No. 1 orNo. 8 or No. 12+1 μL So, FA represented 2 μL single lipid of No. 1 orNo. 8 or No. 12+1 μL FA.

2. Experimental Procedures

1) Conditions of the boiling method: to 100 μL single-strandedHJT-sRNA-m7 solution was added 3 μL single lipid (lipid No. 1 inchloroform solution having a concentration of 5 mg/mL, lipids No. 8/12in chloroform solution having a concentration of 10 mg/mL) or lipidcombination, and heated at 100° C. for 30 min;

MG (monoglyceride): 2 μL lipid No. 34;

DG (diglyceride): 2 μL mixture of equal volume of lipids No. 1/2/3/19/35in chloroform solution;

TG (triglyceride): 2 μL mixture of equal volume of lipids No.6/9/10/13/15/16/18/20/21/22/23/24/25/26/27/28/32/33 in chloroformsolution;

LPC (Lysophosphatidylcholine): 2 μL mixture of equal volume of lipidsNo. 36/37 in chloroform solution;

Cer (Ceramides): 2 μL mixture of equal volume of lipids No. 4/14 inchloroform solution;

So (Sphingoshine): 2 μL mixture of equal volume of lipids No. 17/30/31in chloroform solution;

FA (fatty acid): 2 μL lipid No. 29;

2) Experimental conditions: the final concentration of HJT-sRNA-m7 was200 nM. 24 hours after being added to the cells, the amount ofHJT-sRNA-m7 that entered into the cells was detected by RT-qPCR method(SYBR Green Universal dye method). For the protocols, see “Real-timequantitative PCR detection of intracellular expression of nucleic acidsdelivered by lipid”. All experiments were performed in triplicates.

Conclusion: The results showed that for MRC-5 cells, the mixture (No.1/8/12 mixed in equal volume), No. 1 2 μL+No. 8 1 μL, No. 1 2 μL+No. 121 μL, No. 1 2 μL+MG 1 μL, No. 8 2 μL+MG 1 μL, No. 12 2 μL+No. 8 1 μL,and No. 12 2 μL+So 1 μL, delivered nucleic acid more efficiently.

For Caco-2 cells, the mixtures (No. 1/8/12 in equal volume), No. 1 2μL+No. 8 1 μL, No. 1 2 μL+No. 12 1 μL, No. 1 2 μL+MG 1 μL, No. 8 2 μL+MG1 μL, No. 12 2 μL+No. 8 1 μL, No. 12 2 μL+LPC 1 μL and No. 12 2 μL+So 1μL, delivered nucleic acid more efficiently.

Example 1-3: Delivery of Single-Stranded Nucleic Acid into Cell by LipidCombination

Cell types: A549, MRC-5 and Caco-2 cells.

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group by single lipid and nucleic acid: a mixture of 3 μLsingle lipid (No. 8 or No. 12) and the HJT-sRNA-m7 single-strandednucleic acid solution that was treated by boiling method was added tothe cells and mixed, and the final concentration of RNA was 100 nM;

5) Treatment group by lipid combination PC (No. 12) & PE (No. 8) andnucleic acid mixture: a mixture of 2.25 μL lipid combination (PC (No.12) & PE (No. 8), 2:1, V/V) and the HJT-sRNA-m7 single-stranded nucleicacid solution that was treated by boiling method was added to the cellsand mixed, and the final concentration of RNA was 100 nM;

6) Treatment group by lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2.25 μL lipid combinationPC (No. 12) & PE (No. 8) and 0.75 μL of the following types of lipid,DG, TG, LPC, PC, Cer, So or FA) and the HJT-sRNA-m7 single-strandednucleic acid solution that were treated by boiling method was added tothe cells and mixed, and the final concentration of RNA was 100 nM. InFIG. 18, the mixture treatment group corresponds to the treatment groupswithin the horizontal line above “2.25 μL+0.75 μL”.

2. Experimental Procedures

1) Boiling method conditions: to 100 μL single-stranded HJT-sRNA-m7solution was added single lipid (lipids No. 8/12 in chloroform solutionhaving a concentration of 10 mg/mL) or lipid combination, and heated at100° C. for 30 min;

DG (diglyceride): 0.75 μL mixture of equal volume of lipids No. 1/2 inchloroform solution;

TG (triglyceride): 0.75 μL lipid No. 15 in chloroform solution;

LPC (Lysophosphatidylcholine): 0.75 μL mixture of equal volume of lipidsNo. 36/37 in chloroform solution;

PC (Lysophosphatidylcholine): 0.75 μL lipid No. 12 in chloroformsolution;

Cer (Ceramides): 0.75 μL lipid No. 4 in chloroform solution;

So (Sphingoshine): 0.75 μL lipid No. 31 in chloroform solution;

FA (fatty acid): 0.75 μL lipid No. 29;

2) Experimental conditions: the final concentration of HJT-sRNA-m7 was100 nM. 24 hours after being added to the cells, the amount ofHJT-sRNA-m7 that entered the cells was detected by RT-qPCR method (SYBRGreen Universal dye method). For the protocols, see “Real-timequantitative PCR detection of intracellular expression of nucleic acidsdelivered by lipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that the above single lipids andlipid combinations were effective in delivering nucleic acids into cellsas compared to the free uptake group (see FIG. 18), having the potentialof improving the efficiency of the delivery of nucleic acid drug inclinical settings.

For A549, MRC-5 and Caco-2 cells, 2.25 μL, PC (No. 12) & PE (No. 8)+0.75μL, DG (mixture of equal volume of lipids No. 1/2 in chloroformsolutions) achieved the best efficiency of delivery.

Example 1-4: Delivery of Single-Stranded Nucleic Acid into Cells byLipid Combination

Cell types: A549, MRC-5 and Caco-2 cells.

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of single lipid and nucleic acid: a mixture of 3 μLsingle lipid (No. 8 or No. 12) and the HJT-sRNA-m7 single-strandednucleic acid solution that was treated by boiling method was added tothe cells and mixed, and the final concentration of RNA was 100 nM;

5) Treatment group of lipid combination DG (No. 1) & PE (No. 8) & PC(No. 12) and nucleic acid mixture: a mixture of 3 μL lipid combination(DG (No. 1) & PE (No. 8) & PC (No. 12), 1:1:1, V/V/V) and theHJT-sRNA-m7 single-stranded nucleic acid solution that was treated byboiling method was added to the cells and mixed, and the finalconcentration of RNA was 100 nM;

6) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2 μL lipid combination DG(No. 1) & PE (No. 8) & PC (No. 12) and 1 μL of the following types oflipids, DG, TG, LPC, PC, Cer, So or FA) and the HJT-sRNA-m7single-stranded nucleic acid solution that was treated by boiling methodwas added to the cells and mixed, and the final concentration of RNA was100 nM. In FIG. 19, the mixture treatment groups correspond to thetreatment groups within the horizontal line above 2 μL lipid combinationDG (No. 1) & PE (No. 8) & PC (No. 12))+1 μL.

2. Experimental Procedures

1) Boiling method conditions: to 100 μL single-stranded HJT-sRNA-m7solution was added 3 μL single lipid (lipid No. 1 in chloroform solutionhaving a concentration of 5 mg/mL, lipids No. 8/12 in chloroformsolution having a concentration of 10 mg/mL) or lipid combination, andheated at 100° C. for 30 min;

DG (diglyceride): 1 μL mixture of equal volume of lipids No. 1/2 inchloroform solution;

TG (triglyceride): 1 μL lipid No. 15 in chloroform solution;

LPC (Lysophosphatidylcholine): 1 μL mixture of equal volume of lipidsNo. 36/37 in chloroform solution;

PC (Lysophosphatidylcholine): 1 μL lipid No. 12 in chloroform solution;

Cer (Ceramides): 1 μL lipid No. 4 in chloroform solution;

So (Sphingoshine): 1 μL lipid No. 31 in chloroform solution;

FA (fatty acid): 1 μL lipid No. 29;

2) Experimental conditions: the final concentration of HJT-sRNA-m7 was100 nM. 24 hours after being added to the cells, the amount ofHJT-sRNA-m7 was detected by RT-qPCR method (SYBR Green Universal dyemethod). For the protocols, see “Real-time quantitative PCR detection ofintracellular expression of nucleic acids delivered by lipids”. Allexperiments were performed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells as compared to thefree uptake group (see FIG. 19), having the potential of improving theefficiency of the delivery of nucleic acid drug in clinical settings.

For A549, MRC-5 and Caco-2 cells, 2 μL, DG (No. 1) & PE (No. 8) & PC(No. 12)+1 μL, TG (No. 15) achieved the best efficiency of delivery.

Example 1-5: Delivery of Single-Stranded Nucleic Acid into Cell by LipidCombination

Cell types: A549, MRC-5 and Caco-2 cells.

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of single lipid and nucleic acid: a mixture of 3 μLsingle lipid of No. 8 and the HJT-sRNA-m7 single-stranded nucleic acidsolution that was treated by boiling method was added to the cells andmixed, and the final concentration of RNA was 100 nM;

5) Treatment group of lipid combination PE (No. 8) & MG (No. 34) andnucleic acid mixture: a mixture of 2.25 μL lipid combination (PE (No. 8)& MG (No. 34), 2:1, V/V) and the HJT-sRNA-m7 single-stranded nucleicacid solution that was treated by boiling method was added to the cellsand mixed, and the final concentration of RNA was 100 nM;

6) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2.25 μL lipid combinationPE (No. 8) & MG (No. 34) and 0.75 μL of the following types of lipid,DG, TG, LPC, PC, Cer, So or FA) and the HJT-sRNA-m7 single-strandednucleic acid solution that was treated by boiling method was added tothe cells and mixed, and the final concentration of RNA was 100 nM. InFIG. 20, the mixture treatment group corresponds to the treatment groupswithin the horizontal line above “2.25 μL [lipid combination PE (No. 8)& MG (No. 34)]+0.75 μL”.

2. Experimental Procedures

1) Boiling method conditions: to 100 μL single-stranded HJT-sRNA-m7solution was added single lipid (lipid No. 8 in chloroform solutionhaving a concentration of 10 mg/mL) or lipid combination, and heated at100° C. for 30 min;

DG (diglyceride): 0.75 μL mixture of equal volume of lipids No. 1/2 inchloroform solution;

TG (triglyceride): 0.75 μL lipid No. 15 in chloroform solution;

LPC (Lysophosphatidylcholine): 0.75 μL mixture of equal volume of lipidsNo. 36/37 in chloroform solution;

PC (Lysophosphatidylcholine): 0.75 μL lipid No. 12 in chloroformsolution; Cer (Ceramides): 0.75 μL lipid No. 4 in chloroform solution;

So (Sphingoshine): 0.75 μL lipid No. 31 in chloroform solution;

FA (fatty acid): 0.75 μL lipid No. 29;

2) Experimental conditions: the final concentration of HJT-sRNA-m7 was100 nM.

24 hours after being added to the cells, the amount of HJT-sRNA-m7 thatentered in to cells was detected by RT-qPCR method (SYBR Green Universaldye method). For the protocols, see “Real-time quantitative PCRdetection of intracellular expression of nucleic acids delivered bylipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that the above single lipid and lipidcombinations were effective in delivering nucleic acids into cells ascompared to the free uptake group (see FIG. 20), having the potential ofimproving the efficiency of the delivery of nucleic acid drug inclinical settings.

For A549, MRC-5 and Caco-2 cells, 2.25 μL, PE (No. 8) & MG (No. 34)+0.75μL, So (No. 31) achieved the best efficiency of delivery.

Example 1-6: Delivery of Single-Stranded Nucleic Acid into A549 Cells byLipid Combination

1. Experimental Groups:

1) Naive group: untreated A549 cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of single lipid and nucleic acid: a mixture of 3 μLsingle lipid No. 38 and the HJT-sRNA-m7 single-stranded nucleic acidsolution that was treated by boiling method was added to the cell, andmixed, and the final concentration of RNA was 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2 μL, single lipid No. 38and 1 μL, of the following types of lipid, MG, DG, TG, LPC, PC, PE, Cer,So or FA) and the HJT-sRNA-m7 single-stranded nucleic acid solution thatwas treated by boiling method was added to the cells and mixed, and thefinal concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL single-stranded HJT-sRNA-m7solution was added 3 μL single lipid (lipid No. 38 in chloroformsolution having a concentration of 10 mg/mL) or lipid combination, andheated at 100° C. for 30 min;

MG (monoglyceride): 1 μL lipid No. 34;

DG (diglyceride): 1 μL lipid No. 1 in chloroform solution;

TG (triglyceride): 1 μL lipid No. 15 in chloroform solution;

LPC (Lysophosphatidylcholine): 1 μL lipid No. 37 in chloroform solution;

PC (Lysophosphatidylcholine): 1 μL lipid No. 12 in chloroform solution;

PE (phosphatidylethanolamine): 1 μL lipid No. 8 in chloroform solution;

Cer (Ceramides): 1 μL lipid No. 4 in chloroform solution;

So (Sphingoshine): 1 μL lipid No. 31 in chloroform solution;

FA (fatty acid): 1 μL lipid No. 29 in chloroform solution;

2) Experimental conditions: the final concentration of HJT-sRNA-m7 was100 nM. 24 hours after being added to the cell, the amount ofHJT-sRNA-m7 that entered the cells was detected by RT-qPCR method (SYBRGreen Universal dye method). For the protocols, see “Real-timequantitative PCR detection of intracellular expression of nucleic acidsdelivered by lipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that for A549 cells, the above 2 μLsingle lipid No. 38 and 1 μL LPC (No. 37), TG (No. 15), PC (No. 12), DG(No. 1) were effective in delivering nucleic acids into cells ascompared to the free uptake group (see FIG. 21).

Example 1-7: Delivery of Single-Stranded Nucleic Acid into A549 Cells byLipid Combination

1. Experimental Groups:

1) Naive group: untreated A549 cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination DG (No. 1) & PE (No. 38) & PC(No. 12) and nucleic acid mixture: a mixture of 3 μL lipid combination(DG (No. 1) & PE (No. 38) & PC (No. 12), 1:1:1, V/V/V) and theHJT-sRNA-m7 single-stranded nucleic acid solution that was treated byboiling method was added to the cells and mixed, and the finalconcentration of RNA was 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2 μL lipid combination DG(No. 1) & PE (No. 38) & PC (No. 12) and 1 μL of the following types oflipid, MG, TG, LPC, PE, Cer, So or FA) and the HJT-sRNA-m7single-stranded nucleic acid solution that was treated by boiling methodwas added to the cells and mixed, and the final concentration of RNA was100 nM.

2. Experimental Procedures

1) Boiling method conditions: to 100 μL single-stranded HJT-sRNA-m7solution was added 3 μL lipid combination, and heated at 100° C. for 30min;

MG (monoglyceride): 1 μL lipid No. 34;

TG (triglyceride): 1 μL lipid No. 15 in chloroform solution;

LPC (Lysophosphatidylcholine): 1 μL lipid No. 37 in chloroform solution;

PE (phosphatidylethanolamine): 1 μL lipid No. 8 in chloroform solution;

Cer (Ceramides): 1 μL lipid No. 4 in chloroform solution;

So (Sphingoshine): 1 μL lipid No. 31 in chloroform solution;

FA (fatty acid): 1 μL lipid No. 29 in chloroform solution;

2) Experimental conditions: the final concentration of HJT-sRNA-m7 was100 nM. 24 hours after being added to the cells, the amount ofHJT-sRNA-m7 was detected by RT-qPCR method (SYBR Green Universal dyemethod). For the protocols, see “Real-time quantitative PCR detection ofintracellular expression of nucleic acids delivered by lipids”. Allexperiments were performed in triplicates.

Conclusions: The results indicated that the above 2 μL lipid combinationDG (No. 1) & PE (No. 38) & PC (No. 12) and 1 μL TG (No. 15), Cer (No.4), So (No. 31), FA (No. 29), LPC (No. 37), PE (No. 8) were alleffective in delivering nucleic acids into A549 cells as compared to thefree uptake group (see FIG. 22), having the potential of improving theefficiency of the delivery of nucleic acid drug in clinical settings.

Example 1-8: Delivery of Single-Stranded Nucleic Acid into A549 Cells byLipid Combination

1. Experimental Groups:

1) Naive group: untreated A549 cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination PE (No. 38) & MG (No. 34) andnucleic acid mixture: a mixture of 3 μL lipid combination (PE (No. 38) &MG (No. 34), 2:1, V/V) and the HJT-sRNA-m7 single-stranded nucleic acidsolution that was treated by boiling method was added to the cells andmixed, and the final concentration of RNA was 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2 μL lipid combination PE(No. 38) & MG (No. 34) and 1 μL of the following types of lipid, DG, TG,LPC, PC, PE, Cer, So or FA) and the HJT-sRNA-m7 single-stranded nucleicacid solution that was treated by boiling method was added to the cellsand mixed, and the final concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL single-stranded HJT-sRNA-m7solution was added 3 μL lipid combination, and heated at 100° C. for 30min;

DG (diglyceride): 1 μL lipid No. 1 in chloroform solution;

TG (triglyceride): 1 μL lipid No. 15 in chloroform solution;

LPC (Lysophosphatidylcholine): 1 μL lipid No. 37 in chloroform solution;

PC (phosphatidylcholine): 1 μL lipid No. 12 in chloroform solution;

PE (phosphatidylethanolamine): 1 μL lipid No. 8 in chloroform solution;

Cer (Ceramides): 1 μL lipid No. 4 in chloroform solution;

So (Sphingoshine): 1 μL lipid No. 31 in chloroform solution;

FA (fatty acid): 1 μL lipid No. 29 in chloroform solution;

2) Experimental conditions: the final concentration of HJT-sRNA-m7 was100 nM. 24 hours after being added to the cells, the amount ofHJT-sRNA-m7 was detected by RT-qPCR method (SYBR Green Universal dyemethod). For the protocols, see “Real-time quantitative PCR detection ofintracellular expression of nucleic acids delivered by lipids”. Allexperiments were performed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere all effective in delivering nucleic acids into cells (see FIG. 23),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings, wherein 2 μL lipid combinationPE (No. 38) & MG (No. 34) and 1 μL LPC (No. 37) achieved the bestdelivery effect.

Example 1-9: Delivery of Single-Stranded Nucleic Acid into A549 Cells byLipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination PE (No. 38) & PC (No. 12) andnucleic acid mixture: a mixture of 3 μL lipid combination (PE (No. 38) &PC (No. 12), 2:1, V/V) and the HJT-sRNA-m7 single-stranded nucleic acidsolution that was treated by boiling method was added to the cells, andmixed, and the final concentration of RNA was 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2 μL lipid combination PE(No. 38) & PC (No. 12) and 1 μL of the following types of lipid, MG, DG,TG, LPC, PE, Cer, So or FA) and the HJT-sRNA-m7 single-stranded nucleicacid solution that was treated by boiling method was added to the cells,and mixed, and the final concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL single-stranded HJT-sRNA-m7solution was added 3 μL lipid combination, and heated at 100° C. for 30min;

MG (monoglyceride): 1 μL lipid No. 34;

DG (diglyceride): 1 μL lipid No. 1 in chloroform solution;

TG (triglyceride): 1 μL lipid No. 15 in chloroform solution; LPC(Lysophosphatidylcholine): 1 μL lipid No. 37 in chloroform solution;

PE (phosphatidylethanolamine): 1 μL lipid No. 8 in chloroform solution;

Cer (Ceramides): 1 μL lipid No. 4 in chloroform solution;

So (Sphingoshine): 1 μL lipid No. 31 in chloroform solution;

FA (fatty acid): 1 μL lipid No. 29;

2) Experimental conditions: the final concentration of HJT-sRNA-m7 was100 nM, 24 hours after being added to the cells, the amount ofHJT-sRNA-m7 that entered the cells was detected by RT-qPCR method (SYBRGreen Universal dye method). For the protocols, see “Real-timequantitative PCR detection of intracellular expression of nucleic acidsdelivered by lipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 24),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings, wherein 2 μL lipid combinationPE (No. 38) & PC (No. 12) and 1 μL Cer (No. 4) achieved the best effect.

Example 1-10: Delivery of Single-Stranded Nucleic Acid into A549 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination PE (No. 38) & PC (No. 12) & DG(No. 1) & TG (No. 15) and nucleic acid mixture: a mixture of 3 μL lipidcombination (PE (No. 38) & PC (No. 12) & DG (No. 1) & TG (No. 15),2:2:2:3, V/V/V/V) and the HJT-sRNA-m7 single-stranded nucleic acidsolution that was treated by boiling method was added to the cells, andmixed, and the final concentration of RNA was 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2.2 μL lipid combinationPE (No. 38) & PC (No. 12) & DG (No. 1) & TG (No. 15) and 0.8 μL of thefollowing types of lipid, MG, LPC, Cer,

So or FA) and the HJT-sRNA-m7 single-stranded nucleic acid solution thatwas treated by boiling method was added to the cells, and mixed, and thefinal concentration of RNA was 100 nM.

2. Experimental Procedures

1) Boiling method conditions: to 100 μL single-stranded HJT-sRNA-m7solution was added 3 μL lipid combination, and heated at 100° C. for 30min;

MG (monoglyceride): 0.8 μL lipid No. 34; LPC (Lysophosphatidylcholine):0.8 μL lipid No. 37 in chloroform solution;

Cer (Ceramides): 0.8 μL lipid No. 4 in chloroform solution;

So (Sphingoshine): 0.8 μL lipid No. 31 in chloroform solution;

FA (fatty acid): 0.8 μL lipid No. 29;

2) Experimental conditions: the final concentration of HJT-sRNA-m7 was100 nM, 24 hours after the addition to the cells, the amount ofHJT-sRNA-m7 that entered the cells was detected by RT-qPCR method (SYBRGreen Universal dye method). For the protocols, see “Real-timequantitative PCR detection of intracellular expression of nucleic acidsdelivered by lipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 25),wherein 2.2 μL lipid combination PE (No. 38) & PC (No. 12) & DG (No. 1)& TG (No. 15), 2.2 μL lipid combination PE (No. 38) & PC (No. 12) & DG(No. 1) & TG (No. 15) and 0.8 μL LPC (No. 37) or So (No. 31) achievedrelative better efficiency of delivery.

Example 1-11: Delivery of Single-Stranded Nucleic Acid into A549 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination PE (No. 38) & MG (No. 34) & LPC(No. 37) and nucleic acid mixture: a mixture of 3 μL lipid combination(PE (No. 38) & MG (No. 34) & LPC (No. 37), 4:2:3, V/V/V) and theHJT-sRNA-m7 single-stranded nucleic acid solution that was treated byboiling method was added to the cells, and mixed, and the finalconcentration of RNA was 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2.2 μL lipid combinationPE (No. 38) & MG (No. 34) & LPC (No. 37) and 0.8 μL of the followingtypes of lipid, DG, TG, PC, Cer, or So) and the HJT-sRNA-m7single-stranded nucleic acid solution that was treated by boiling methodwas added to the cells, and mixed, and the final concentration of RNAwas 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL single-stranded HJT-sRNA-m7solution was added 3 μL lipid combination, and heated at 100° C. for 30min;

DG (diglyceride): 0.8 μL lipid No. 1 in chloroform solution;

TG (triglyceride): 0.8 μL lipid No. 15 in chloroform solution;

PC (phosphatidylcholine): 0.8 μL lipid No. 12 in chloroform solution;

Cer (Ceramides): 0.8 μL lipid No. 4 in chloroform solution;

So (Sphingoshine): 0.8 μL lipid No. 31 in chloroform solution; 2)Experimental conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method (SYBR GreenUniversal dye method). For the protocols, see “Real-time quantitativePCR detection of intracellular expression of nucleic acids delivered bylipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 26),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings.

Example 1-12: Delivery of Single-Stranded Nucleic Acid into A549 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination PE (No. 38) & PC (No. 12) & Cer(No. 4) and nucleic acid mixture: a mixture of 3 μL lipid combination(PE (No. 38) & PC (No. 12) & Cer (No. 4), 4:2:3, V/V/V) and theHJT-sRNA-m7 single-stranded nucleic acid solution that was treated byboiling method was added to the cells, and mixed, and the finalconcentration of RNA was 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2.2 μL lipid combinationPE (No. 38) & PC (No. 12) & Cer (No. 4) and 0.8 μL of the followingtypes of lipid, MG, DG, TG, LPC, So or FA) and the HJT-sRNA-m7single-stranded nucleic acid solution that was treated by boiling methodwas added to the cells, and mixed, and the final concentration of RNAwas 100 nM.

2. Experimental Procedures

1) Boiling method conditions: to 100 μL single-stranded HJT-sRNA-m7solution was added 3 μL lipid combination, and heated at 100° C. for 30min;

MG (monoglyceride): 0.8 μL lipid No. 34;

DG (diglyceride): 0.8 μL lipid No. 1 in chloroform solution;

TG (triglyceride): 0.8 μL lipid No. 15 in chloroform solution; LPC(lysophosphatidylcholine): 0.8 μL lipid No. 37 in chloroform solution;

So (Sphingoshine): 0.8 μL lipid No. 31 in chloroform solution;

FA (fatty acid): 0.8 μL, lipid No. 29 in chloroform solution;

2) Experimental conditions: the final concentration of HJT-sRNA-m7 was100 nM, 24 hours after the addition to the cells, the amount ofHJT-sRNA-m7 that entered the cells was detected by RT-qPCR method (SYBRGreen Universal dye method). For the protocols, see “Real-timequantitative PCR detection of intracellular expression of nucleic acidsdelivered by lipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 27),wherein 2.2 μL, lipid combination PE (No. 38) & PC (No. 12) & Cer (No.4) and 0.8 μL, FA (No. 29) achieved the best efficiency of delivery.

Example 1-13: Delivery of Single-Stranded Nucleic Acid into A549 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination PE (No. 38) & PC (No. 12) & Cer(No. 4) & FA (No. 29) and nucleic acid mixture: a mixture of 3 μL lipidcombination (PE (No. 38) & PC (No. 12) & Cer (No. 4) & FA (No. 29),44:22:33:36, V/V/V/V) and the HJT-sRNA-m7 single-stranded nucleic acidsolution that was treated by boiling method was added to the cells, andmixed, and the final concentration of RNA was 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of PE (No. 38) & PC (No. 12)& Cer (No. 4) & FA (No. 29) and 1 μL of the following types of lipid)and the HJT-sRNA-m7 single-stranded nucleic acid solution that wastreated by boiling method was added to the cells, and mixed, and thefinal concentration of RNA was 100 nM.

2. Experimental Procedures

1) Boiling method conditions: to 100 μL single-stranded HJT-sRNA-m7solution was added 3 μL lipid combination, and heated at 100° C. for 30min;

MG (monoglyceride): 1 μL lipid No. 34;

DG (diglyceride): 1 μL lipid No. 1 in chloroform solution;

TG (triglyceride): 1 μL lipid No. 15 in chloroform solution; LPC(lysophosphatidylcholine): 1 μL lipid No. 37 in chloroform solution;

So (Sphingoshine): 1 μL lipid No. 31 in chloroform solution;

2) Experimental conditions: the final concentration of HJT-sRNA-m7 was100 nM, 24 hours after the addition to the cells, the amount ofHJT-sRNA-m7 that entered the cells was detected by RT-qPCR method (SYBRGreen Universal dye method). For the protocols, see “Real-timequantitative PCR detection of intracellular expression of nucleic acidsdelivered by lipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 28),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings.

Example 1-14: Delivery of Single-Stranded Nucleic Acid into A549 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination PE (No. 38) & PC (No. 12) & So(No. 31) and nucleic acid mixture: a mixture of 3 μL lipid combination(PE (No. 38) & PC (No. 12) & So (No. 31), 2:1:3, V/V/V) and theHJT-sRNA-m7 single-stranded nucleic acid solution that was treated byboiling method was added to the cells, and mixed, and the finalconcentration of RNA was 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2 μL PE (No. 38) & PC (No.12) & So (No. 31) and 1 μL of the following types of lipid, MG, DG, TG,LPC, Cer or FA) and the HJT-sRNA-m7 single-stranded nucleic acidsolution that was treated by boiling method was added to the cells, andmixed, and the final concentration of RNA was 100 nM.

2. Experimental Procedures

1) Boiling method conditions: to 100 μL Single-stranded HJT-sRNA-m7solution was added 3 μL lipid combination, and heated at 100° C. for 30min;

DG (diglyceride): 1 μL lipid No. 1 in chloroform solution;

TG (triglyceride): 1 μL lipid No. 15 in chloroform solution;

PC (phosphatidylcholine): 1 μL lipid No. 12 in chloroform solution;

Cer (Ceramides): 1 μL lipid No. 4 in chloroform solution;

FA (fatty acid): 1 μL lipid No. 29 in chloroform solution;

2) Experimental conditions: the final concentration of HJT-sRNA-m7 was100 nM, 24 hours after the addition to the cells, the amount ofHJT-sRNA-m7 that entered the cells was detected by RT-qPCR method (SYBRGreen Universal dye method). For the protocols, see “Real-timequantitative PCR detection of intracellular expression of nucleic acidsdelivered by lipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 29),wherein 2 μL lipid combination PE (No. 38) & PC (No. 12) & So (No. 31)and 1 μL FA (No. 29) achieved the best delivery effect.

Example 1-15: Delivery of Single-Stranded Nucleic Acid into A549 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination PE (No. 38) & MG (No. 34) & LPC(No. 37) & So (No. 31) and nucleic acid mixture: a mixture of 3 μL lipidcombination (PE (No. 38) & MG (No. 34) & LPC (No. 37) & So (No. 31),44:22:33:36, V/V/V/V) and the HJT-sRNA-m7 single-stranded nucleic acidsolution that was treated by boiling method was added to the cells, andmixed, and the final concentration of RNA was 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2 μL PE (No. 38) & MG (No.34) & LPC (No. 37) & So (No. 31) and 1 μL of the following types oflipid, DG, TG, PC, Cer or FA) and the HJT-sRNA-m7 single-strandednucleic acid solution that was treated by boiling method was added tothe cells, and mixed, and the final concentration of RNA was 100 nM.

2. Experimental Procedures

1) Boiling method conditions: to 100 μL single-stranded HJT-sRNA-m7solution was added 3 μL lipid combination, and heated at 100° C. for 30min;

DG (diglyceride): 1 μL lipid No. 1 in chloroform solution;

TG (triglyceride): 1 μL lipid No. 15 in chloroform solution;

PC (phosphatidylcholine): 1 μL lipid No. 12 in chloroform solution;

Cer (Ceramides): 1 μL lipid No. 4 in chloroform solution;

FA (fatty acid): 1 μL lipid No. 29 in chloroform solution;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method (SYBR GreenUniversal dye method). For the protocols, see “Real-time quantitativePCR detection of intracellular expression of nucleic acids delivered bylipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that as compared to the free uptakegroup, the addition of 1 μL DG (No. 1), TG (No. 15), PC (No. 12), Cer(No. 4) or FA (No. 29) to 2 μL PE (No. 38) & MG (No. 34) & LPC (No. 37)& So (No. 31), could efficiently deliver nucleic acids into cells (seeFIG. 30), having the potential of improving the efficiency of thedelivery of nucleic acid drug in clinical settings. The addition of 1 μLPC (No. 12) achieved the best efficiency in nucleic acid delivery andcould enhance the efficiency of delivery.

Example 1-16: Delivery of Single-Stranded Nucleic Acid into A549 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination PE (No. 38) & LPC (No. 37) andnucleic acid mixture: a mixture of 3 μL lipid combination (PE (No. 38) &LPC (No. 37), 2:1, V/V) and the HJT-sRNA-m7 single-stranded nucleic acidsolution that was treated by boiling method was added to the cells, andmixed, and the final concentration of RNA was 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2 μL PE (No. 38) & LPC(No. 37) and 1 μL of the following types of lipid, MG, DG, TG, PC, Cer,So or FA) and the HJT-sRNA-m7 single-stranded nucleic acid solution thatwas treated by boiling method was added to the cells, and mixed, and thefinal concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL single-stranded HJT-sRNA-m7solution was added 3 μL lipid combination, and heated at 100° C. for 30min;

MG (monoglyceride): 1 μL lipid No. 34;

DG (diglyceride): 1 μL lipid No. 1 in chloroform solution;

TG (triglyceride): 1 μL lipid No. 15 in chloroform solution;

PC (phosphatidylcholine): 1 μL lipid No. 12 in chloroform solution;

Cer (Ceramides): 1 μL lipid No. 4 in chloroform solution;

So (Sphingoshine): 1 μL lipid No. 31 in chloroform solution;

FA (fatty acid): 1 μL lipid No. 29 in chloroform solution;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method (SYBR GreenUniversal dye method). For the protocols, see “Real-time quantitativePCR detection of intracellular expression of nucleic acids delivered bylipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that as compared to the free uptakegroup, the above lipid combinations were effective in delivering nucleicacids into cells (see FIG. 31), having the potential of improving theefficiency of the delivery of nucleic acid drug in clinical settings.The addition of 1 μL TG (No. 15) to 2 μL lipid combination PE (No. 38) &LPC (No. 37) achieved the best effect in nucleic acid delivery.

Example 1-17: Delivery of Single-Stranded Nucleic Acid into A549 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: single-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination PE (No. 38) & LPC (No. 37) & TG(No. 15) and nucleic acid mixture: a mixture of 3 μL lipid combination(PE (No. 38) & LPC (No. 37) & TG (No. 15), 32:8:5, V/V/V) and theHJT-sRNA-m7 single-stranded nucleic acid solution that was treated byboiling method was added to the cells, and mixed, and the finalconcentration of RNA was 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2 μL PE (No. 38) & LPC(No. 37) & TG (No. 15) and μL of the following types of lipid, MG, DG,PC, Cer, So or FA) and the HJT-sRNA-m7 single-stranded nucleic acidsolution that was treated by boiling method was added to the cells, andmixed, and the final concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL single-stranded HJT-sRNA-m7solution was added 3 μL lipid combination, and heated at 100° C. for 30min;

MG (monoglyceride): 1 μL lipid No. 34;

DG (diglyceride): 1 μL lipid No. 1 in chloroform solution;

PC (phosphatidylcholine): 1 μL lipid No. 12 in chloroform solution;

Cer (Ceramides): 1 μL lipid No. 4 in chloroform solution;

So (Sphingoshine): 1 μL lipid No. 31 in chloroform solution;

FA (fatty acid): 1 μL lipid No. 29 in chloroform solution;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method (SYBR GreenUniversal dye method). For the protocols, see “Real-time quantitativePCR detection of intracellular expression of nucleic acids delivered bylipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 32),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings. The lipid combination PE (No.38) & LPC (No. 37) & TG (No. 15) efficiently delivered nucleic acidsinto cells. Further addition of other types of lipid to the lipidcombination PE (No. 38) & LPC (No. 37) & TG (No. 15) did not enhancethis effect.

Example 2-1: Delivery of Double-Stranded Nucleic Acid into MRC-5 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of single lipid and nucleic acid: a mixture of 3 μLsingle lipid No. 38 and the HJT-sRNA-m7 double-stranded nucleic acidsolution that was treated by boiling method was added to the cells, andmixed, and the final concentration of RNA was 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2 μL single lipid No. 38and 1 μL lipid No. 8, 1, 2, 11, 12, 34, 37, 4, 30, 31, 29, 32, 1+2(mixed in equal volume) or 11+12 (mixed in equal volume) in chloroformsolution) and the HJT-sRNA-m7 double-stranded nucleic acid solution thatwas treated by boiling method was added to the cells, and mixed, and thefinal concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added 3 μL single lipid (lipid No. 38 in chloroformsolution having a concentration of 10 mg/mL) or lipid combination, andheated at 100° C. for 30 min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method (SYBR GreenUniversal dye method). For the protocols, see “Real-time quantitativePCR detection of intracellular expression of nucleic acids delivered bylipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that the above single lipids andlipid combinations were effective in delivering nucleic acids into cells(see FIG. 33), having the potential of improving the efficiency of thedelivery of nucleic acid drug in clinical settings. The single lipid No.38 effectively delivered nucleic acids into MRC-5 cells, showing aneffect close to the transfection reagent RNAiMAX. The addition of otherlipids to it did not further enhance the effect.

Example 2-2: Delivery of Double-Stranded Nucleic Acid into MRC-5 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination (No. 38 & No. 37, 2:1, V/V) andnucleic acid: a mixture of 3 μL lipid combination and the HJT-sRNA-m7double-stranded nucleic acid solution that was treated by boiling methodwas added to the cells, and mixed, and the final concentration of RNAwas 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2 μL lipid combination No.38 & No. 37 and 1 μL lipid No. 8, 1, 2, 11, 12, 34, 37, 4, 30, 31, 29,32, 1+2 (mixed in equal volume) or 11+12 (mixed in equal volume) inchloroform solution) and the HJT-sRNA-m7 double-stranded nucleic acidsolution that was treated by boiling method was added to the cells, andmixed, and the final concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added to 3 μL lipid combination, and heated at 100° C. for30 min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method (SYBR GreenUniversal dye method). For the protocols, see “Real-time quantitativePCR detection of intracellular expression of nucleic acids delivered bylipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that the above single lipids andlipid combinations were effective in delivering nucleic acids into cells(see FIG. 34), having the potential of improving the efficiency of thedelivery of nucleic acid drug in clinical settings. Lipid No. 38 & No.37 mixture efficiently delivered nucleic acids into MRC-5 cells. Toaddition of 1 μL lipids, except No. 11 and 34, to 2 μL No. 38 & No. 37mixture could enhance this effect. In addition, unexpectedly, theaddition of 1 μL lipid No. 32 to 2 μL No. 38 & No 37 mixture achievedthe best effect, even better than the effect of RNAiMAX.

Example 2-3: Delivery of Double-Stranded Nucleic Acid into A549 Cells byLipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination (PE (No. 38) & PC (No. 12) & Cer(No. 4)) and nucleic acid: a mixture of 3 μL lipid combination (PE (No.38) & PC (No. 12) & Cer (No. 4), 4:2:3, V/V/V) and the HJT-sRNA-m7double-stranded nucleic acid solution that was treated by boiling methodwas added to the cells, and mixed, and the final concentration of RNAwas 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2.5 μL PE (No. 38) & PC(No. 12) & Cer (No. 4) and 0.5 μL lipids (DG (No. 2), TG (No. 6), So(No. 17), FA (No. 29), MG (No. 34) and LPC (No. 37)) and the HJT-sRNA-m7double-stranded nucleic acid solution that was treated by boiling methodwas added to the cells, and mixed, and the final concentration of RNAwas 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL HJT-sRNA-m7 double-strandedsolution was added 3 μL lipid combination, and heated at 100° C. for 30min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method (SYBR GreenUniversal dye method). For the protocols, see “Real-time quantitativePCR detection of intracellular expression of nucleic acids delivered bylipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that the above single lipids andlipid combinations were effective in delivering nucleic acids into cells(see FIG. 35), having the potential of improving the efficiency of thedelivery of nucleic acid drug in clinical settings. The addition of ⅕LPC (No. 37) to PE (No. 38) & PC (No. 12) & Cer (No. 4) mixture couldsignificantly enhance the effect in delivery of the nucleic acid. Inaddition, the addition of DG (No. 2) and TG (No. 16) could also furtherenhance the effect in delivery.

Example 2-4: Delivery of Double-Stranded Nucleic Acid into A549 Cells byLipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination (PE (No. 38) & DG (No. 2)) andnucleic acid: a mixture of 3 μL lipid combination (PE (No. 38) & DG (No.2), 2:1, V/V) and the HJT-sRNA-m7 double-stranded nucleic acid solutionthat was treated by boiling method was added to the cells, and mixed,and the final concentration of RNA was 100 nM;

5) Treatment group of lipid combination and nucleic acid mixture: amixture of 3 μL lipid combination (mixture of 2 μL PE (No. 38) & DG (No.2) mixture and 1 μL other lipid of No. 37, 31, 29, 34, 12 or 4) and theHJT-sRNA-m7 double-stranded nucleic acid solution that was treated byboiling method was added to the cells, and mixed, and the finalconcentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added 3 μL lipid combination, and heated at 100° C. for 30min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method (SYBR GreenUniversal dye method). For the protocols, see “Real-time quantitativePCR detection of intracellular expression of nucleic acids delivered bylipids”. All experiments were performed in triplicates.

Conclusions: The results indicated that the above single lipids andlipid combinations were effective in delivering nucleic acids into cells(see FIG. 36), having the potential of improving the efficiency of thedelivery of nucleic acid drug in clinical settings. Lipid combination (2μL PE (No. 38) & DG (No. 2) mixture) could effectively deliver thedouble stranded nucleic acid into the A549 cells. As compared with thislipid combination, the lipid combination of 2 μL PE (No. 38) & DG (No.2) and No. 37, 31, 12 or 4 mixed at a ratio of 2:1 could enhance theefficiency of delivery.

Example 2-5: Delivery of Double-Stranded Nucleic Acid into A549 Cells byLipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) A mixture of lipid combination (PE (No. 38) & LPC (No. 37), 4:1, V/V)and the HJT-sRNA-m7 double-stranded nucleic acid solution that wastreated by boiling method was added to the cells, and mixed, and thefinal concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added 3 μL lipid combination, and heated at 70° C. for 30min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method. For theprotocols, see “Real-time quantitative PCR detection of intracellularexpression of nucleic acids delivered by lipids”. All experiments wereperformed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 37),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings, with an effect close to thetransfection reagent RNAiMAX.

Example 2-6: Delivery of Double-Stranded Nucleic Acid into A549 Cells byLipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) A mixture of lipid combination (PE (No. 38) & PC (No. 12), 4:1, V/V)and the HJT-sRNA-m7 double-stranded nucleic acid solution that wastreated by boiling method was added to the cells, and mixed, and thefinal concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added 3 μL, lipid combination, and heated at 70° C. for 30min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method. For theprotocols, see “Real-time quantitative PCR detection of intracellularexpression of nucleic acids delivered by lipids”. All experiments wereperformed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 38),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings. The effect is better than or thesame as that of RNAiMAX.

Example 2-7: Delivery of Double-Stranded Nucleic Acid into A549 Cells byLipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) A mixture of lipid combination (PE (No. 38) & PC (No. 12) & DG (No.2), 4:1:5, V/V/V) and the double-stranded HJT-sRNA-m7 nucleic acidsolution that was treated by boiling method was added to the cells, andmixed, and the final concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added 2 μL lipid combination, and heated at 80° C. for 30min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method. For theprotocols, see “Real-time quantitative PCR detection of intracellularexpression of nucleic acids delivered by lipids”. All experiments wereperformed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 39),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings. Lipid combination (PE (No. 38) &PC (No. 12) & DG (No. 2), 4:1:5, V/V/V) showed better effect in thedelivery of double-stranded nucleic acid into A549 cells than RNAiMAX.

Example 2-8: Delivery of Double-Stranded Nucleic Acid into A549 Cells byLipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) A mixture of lipid combination (PE (No. 38) & LPC (No. 37) & DG (No.2), 32:8:5, V/V/V) and the HJT-sRNA-m7 double-stranded nucleic acidsolution that was treated by boiling method was added to the cells, andmixed, and the final concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added 2 μL lipid combination, and heated at 80° C. for 30min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method. For theprotocols, see “Real-time quantitative PCR detection of intracellularexpression of nucleic acids delivered by lipids”. All experiments wereperformed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 40),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings. The effect was similar to thatof RNAiMAX.

Example 2-9: Delivery of Double-Stranded Nucleic Acid into A549 Cells byLipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) A mixture of lipid combination (PE (No. 8) & PC (No. 12), 1:2, V/V)and the HJT-sRNA-m7 double-stranded nucleic acid solution that wastreated by boiling method was added to the cells, and mixed, and thefinal concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added to 2 μL lipid combination, and heated at 80° C. for30 min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method. For theprotocols, see “Real-time quantitative PCR detection of intracellularexpression of nucleic acids delivered by lipids”. All experiments wereperformed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 41),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings. Lipid combination (PE (No. 8) &PC (No. 12), 1:2, V/V) showed better effect in the delivery ofdouble-stranded nucleic acid into A549 cells than RNAiMAX.

Example 2-10: Delivery of Double-Stranded Nucleic Acid into A549 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) A mixture of lipid combination (PE (No. 8) & LPC (No. 37), 4:1, V/V)and the HJT-sRNA-m7 double-stranded nucleic acid solution that wastreated by boiling method was added to the cells, and mixed, and thefinal concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added 2 μL lipid combination, and heated at 80° C. for 30min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method. For theprotocols, see “Real-time quantitative PCR detection of intracellularexpression of nucleic acids delivered by lipids”. All experiments wereperformed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 42),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings.

Example 2-11: Delivery of Double-Stranded Nucleic Acid into MRC-5 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) A mixture of Lipid combination (PE (No. 8) & PC (No. 12), 1:2, V/V)and the HJT-sRNA-m7 double-stranded nucleic acid solution that wastreated by boiling method was added to the cells, and mixed, and thefinal concentration of RNA was 100 nM;

5) Treatment group of lipid combination and double-stranded HJT-sRNA-m7mixture: a mixture of 3 μL lipid combination (mixture of 2 μL PE (No. 8)& PC (No. 12) and 1 μL other type of lipids (MG (No. 34), DG (No. 2), TG(No. 32), LPC (No. 37), PC (No. 11), PE (No. 38), Cer (No. 4), So (No.31) or FA (No. 29)) and the double-stranded HJT-sRNA-m7 nucleic acidsolution that was treated by boiling method was added to the cells, andmixed, and the final concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added to 3 μL, lipid combination, and heated at 80° C. for30 min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method. For theprotocols, see “Real-time quantitative PCR detection of intracellularexpression of nucleic acids delivered by lipids”. All experiments wereperformed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 43),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings. PE (No. 8) & PC (No. 12) couldeffectively deliver nucleic acids into cells with significantly bettereffect than RNAiMAX. Compared to PE (No. 8) & PC (No. 12), a mixture ofPE (No. 8) & PC (No. 12) and Cer (No. 4) or PE (No. 38) at a ratio of2:1 could enhance this effect.

Example 2-12: Delivery of Double-Stranded Nucleic Acid into MRC-5 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) A mixture of lipid combination (PE (No. 8) & PC (No. 12) &DG (No. 2),8:16:3, V/V/V) and the HJT-sRNA-m7 double-stranded nucleic acid solutionthat was treated by boiling method was added to the cells, and mixed,and the final concentration of RNA was 100 nM;

5). Experimental procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added 2 μL, lipid combination, and heated at 80° C. for 30min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method. For theprotocols, see “Real-time quantitative PCR detection of intracellularexpression of nucleic acids delivered by lipids”. All experiments wereperformed in triplicates.

Conclusions: The results indicated that as compared with the free uptakegroup and RNAiMAX group, the lipid combination (PE (No. 8) & PC (No. 12)&DG (No. 2), 8:16:3, V/V/V) showed better effect in delivery thanRNAiMAX (see FIG. 44), having the potential of improving the efficiencyof the delivery of nucleic acid drug in clinical settings.

Example 2-13: Delivery of Double-Stranded Nucleic Acid into MRC-5 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) A mixture of lipid combination and the HJT-sRNA-m7 double-strandednucleic acid solution that was treated by boiling method was added tothe cells, and mixed, and the final concentration of RNA was 100 nM;

Mixture 1: PE (No. 8):LPC (No. 37):TG (No. 32)-4:1:2

Mixture 2: PE (No. 8):LPC(No. 37):DG (No. 2)-4:1:2

Mixture 3: PE (No. 8):PC (No. 12):So (No. 31):FA (No. 29)-1:2:1:1

5). Experimental procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added 2.5 μL lipid combination, and heated at 90° C. for 15min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method. For theprotocols, see “Real-time quantitative PCR detection of intracellularexpression of nucleic acids delivered by lipids”. All experiments wereperformed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 45),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings. As compared with RNAiMAX group,mixture 1: PE (No. 8):LPC (No. 37):TG (No. 32)-4:1:2, and mixture 2: PE(No. 8):LPC(No. 37):DG (No. 2)-4:1:2 showed comparable effect indelivery, whereas mixture 3: PE (No. 8):PC (No. 12):So (No. 31):FA (No.29)-1:2:1:1 showed better effect.

Example 2-14: Delivery of Double-Stranded Nucleic Acid into MRC-5 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: referred to untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination mixture and double-strandedHJT-sRNA-m7 mixture: a mixture of 3 μL lipid combination (PE (No. 8):PC(No. 12):So (No. 31):FA (No. 29)-1:2:1:1) and the HJT-sRNA-m7double-stranded nucleic acid solution that was treated by boiling methodwas added to the cells, and mixed, and the final concentration of RNAwas 100 nM;

5) Treatment group of lipid combination and double-stranded HJT-sRNA-m7mixture: a mixture of 3 μL lipid combination (mixture of 2 μL, lipidcombination mix and 1 μL, other type of lipid shown in FIG. 46, i.e.lipids No. 34, 2, 32, 11, 37, 38 or 4) and the HJT-sRNA-m7double-stranded nucleic acid solution that was treated by boiling methodwas added to the cells, and mixed, and the final concentration of RNAwas 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added 3 μL, lipid combination, and heated at 90° C. for 15min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method. For theprotocols, see “Real-time quantitative PCR detection of intracellularexpression of nucleic acids delivered by lipids”. All experiments wereperformed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective in delivering nucleic acids into cells (see FIG. 46),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings. The mixture (PE (No. 8):PC (No.12):So (No. 31):FA (No. 29)-1:2:1:1) showed better effect in deliverythan RNAiMAX. Compared to mixture (PE (No. 8):PC (No. 12):So (No. 31):FA(No. 29)-1:2:1:1), the addition of mixture PE (No. 8):PC (No. 12):So(No. 31):FA (No. 29)-1:2:1:1) to lipids No. 2, 38 or 4 at a ratio of 2:1could enhance the delivery effect.

Example 2-15: Delivery of Double-Stranded Nucleic Acid into MRC-5 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) A mixture of lipid combination (PE (No. 8) & So (No. 31), 6:1, V/V)and the HJT-sRNA-m7 double-stranded nucleic acid solution that wastreated by boiling method was added to the cells, and mixed, and thefinal concentration of RNA was 100 nM;

5). Experimental procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added 2 μL, lipid combination, and heated at 90° C. for 15min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 24 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method. For theprotocols, see “Real-time quantitative PCR detection of intracellularexpression of nucleic acids delivered by lipids”. All experiments wereperformed in triplicates.

Conclusions: The results indicated that the lipid combination (PE (No.8) & So (No. 31), 6:1, V/V) showed better effect in delivery thanRNAiMAX (see FIG. 47), having the potential of improving the efficiencyof the delivery of nucleic acid drug in clinical settings.

Example 2-16: Delivery of Double-Stranded Nucleic Acid into MRC-5 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination (PE (No. 8) & So (No. 31), 4:1,V/V) and the HJT-sRNA-m7 mixture: a mixture of 2 μL, the lipidcombination and the HJT-sRNA-m7 double-stranded nucleic acid solutionthat was treated by boiling method was added to the cells, and mixed,and the final concentration of RNA was 100 nM;

5) Treatment group of lipid combination and double-stranded HJT-sRNA-m7mixture: a mixture of lipid combination (a mixture of PE (No. 8) & So(No. 31), 4:1, V/V) and other types of lipid (MG (No. 34), DG (No. 2),LPC (No. 37), PC (No. 12), PC (No. 11), Cer (No. 4), FA (No. 29) or TG(No. 32), 12:3:5, V/V, FIG. 48) and the HJT-sRNA-m7 double-strandednucleic acid solution that was treated by boiling method was added tothe cells, and mixed, and the final concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added 2 μL lipid combination, and heated at 90° C. for 15min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 12 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method. For theprotocols, see “Real-time quantitative PCR detection of intracellularexpression of nucleic acids delivered by lipids”. All experiments wereperformed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective for delivering nucleic acids into cells (see FIG. 48),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings. PE (No. 8):So (No. 31) (4:1,V/V) could effectively deliver nucleic acids into cells with an effectclose to RNAiMAX. Compared to PE (No. 8):So (No. 31), the mixture of PE(No. 8):So (No. 31) and MG (No. 34), DG (No. 2), PC (No. 12), PC (No.11), or TG (No. 32) at a ratio of 12:3:5 could enhance the effect indelivery of nucleic acid, and PE (No. 8):So (No. 31):PC (No. 11) showedthe best effect, significantly better than RNAiMAX.

Example 2-17: Delivery of Double-Stranded Nucleic Acid into MRC-5 Cellsby Lipid Combination

1. Experimental Groups:

1) Naive group: untreated cell;

2) RNAiMAX treatment group: 2 μl RNAiMAX transfection reagent anddouble-stranded HJT-sRNA-m7 solution were diluted in 100 μl opti-MEMmedium, respectively, and then the two were mixed, allowed to stand for15 min, added into cells, and then mixed. The final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

3) Free uptake group: double-stranded HJT-sRNA-m7 solution was directlyadded (the final concentration was 100 nM);

4) Treatment group of lipid combination (PE (No. 8):Cer (No. 4), 4:1,V/V) and the HJT-sRNA-m7 mixture: a mixture of 2 μL lipid combinationand the HJT-sRNA-m7 double-stranded nucleic acid solution that wastreated by boiling method was added to the cells, and mixed, and thefinal concentration of RNA was 100 nM;

5) Treatment group of lipid combination and double-stranded HJT-sRNA-m7mixture: a mixture of lipid combination (mixture of PE (No. 8):Cer (No.4) and other types of lipids MG (No. 34), DG (No. 2), LPC (No. 37), PC(No. 12), PC (No. 31), FA (No. 29) or TG (No. 32), 12:3:5, V/V, FIG. 49)and the double-stranded HJT-sRNA-m7 nucleic acid solution that wastreated by boiling method was added to the cells, and mixed, and thefinal concentration of RNA was 100 nM;

2. Experimental Procedures

1) Boiling method conditions: to 100 μL double-stranded HJT-sRNA-m7solution was added 2 μL lipid combination, and heated at 90° C. for 15min;

2) Experiment conditions: the final concentration of HJT-sRNA-m7 was 100nM, 12 hours after the addition to the cells, the amount of HJT-sRNA-m7that entered the cells was detected by RT-qPCR method. For theprotocols, see “Real-time quantitative PCR detection of intracellularexpression of nucleic acids delivered by lipids”. All experiments wereperformed in triplicates.

Conclusions: The results indicated that the above lipid combinationswere effective for delivering nucleic acids into cells (see FIG. 49),having the potential of improving the efficiency of the delivery ofnucleic acid drug in clinical settings. Lipid combination PE (No. 8):Cer(No. 4) could effectively deliver nucleic acids into cells with aneffect close to RNAiMAX. Compared to PE (No. 8): So (No. 31), themixture of PE (No. 8): So (No. 31) and DG (No. 2), FA (No. 29) or TG(No. 32) at a ratio of 12:3:5 could enhance the effect in delivery ofnucleic acid, and FA (No. 29) could significantly improve the effect(significantly better than RNAiMAX) of PE (No. 8):So (No. 31) indelivery.

Example 3: Lipid Combination Promotes Nucleic Acid Entry into the LungThrough Digestive Tract

The lipid combination was as follow:

Lipids PE (No. 38) & LPC (No. 37) & TG (No. 32), 4:2:3, V/V/V

1. Preparation of Lipid Nucleic Acid Mixture:

Method: boiling method

To 400 μL of HJT-sRNA-m7 (5 nmol) single-stranded RNA in DEPC-treatedaqueous solution was added 9 μL or 18 μL lipid combination (lipid PE(No. 38) & LPC (No. 37) & TG (No. 32), 4:2:3, V/V/V), mixed and heatedat 100° C. for 30 min.

2. Delivery Experiment of Nucleic Acid Via Digestive Tract

RNA was administered via gavage to 6-8 weeks old male C57 mice:HJT-sRNA-m7 in aqueous solution or a mixture solution of lipid andHJT-sRNA-m7 was administered via gavage needle, 400 μL/animal(HJT-sRNA-m7, 5 nmol/animal). Groups were as follows:

(1) Control group (naive group): mice that did not receive anytreatment;

(2) Negative control group (lipid group): administration of 9 μL lipidcombinations (lipid PE (No. 38) & LPC (No. 37) & TG (No. 32), 4:2:3,V/V/V) via gavage;

(3) Free uptake group: direct administration of single-strandedHJT-sRNA-m7 RNA via gavage;

(4) Lipid and nucleic acid mixture group: administration of a mixture oflipid combination and single-stranded HJT-sRNA-m7 RNA via gavage.

3 hours after administration via gavage, the mouse whole lung was lysedwith 3 mL TRIzol, the total RNA was extracted and the abundance ofHJT-sRNA-m7 was detected by RT-qPCR.

Conclusion: As shown in FIG. 50, 9 μL or 18 μL lipid combination (lipidPE (No. 38) & LPC (No. 37) & TG (No. 32), 4:2:3, V/V/V) significantlypromoted the entry of small fragments of nucleic acids into lung tissue(* indicating a P value of less than 0.05) as compared to the freeuptake group. With this (non-invasive) administration via gavage, thelipid combination (lipid PE (No. 38) & LPC (No. 37) & TG (No. 32),4:2:3, V/V/V) could promote small fragments of nucleic acids enteringthe lung tissue, which could be used as a means of nucleic acid drugdelivery.

Example 4: Function Experiments of Delivery of Double-Stranded NucleicAcid into Cells Mediated by Chinese Traditional Medicine-Derived LipidMixture

1. No. 8 (PE):No. 12 (PC) (v:v=1:2) lipid mixture mediated the entry ofnucleic acids into cells to function

Experimental method: Western blot, see above “Western blot detection ofprotein expression level”.

1) No. 8 (PE):No. 12 (PC) (v:v=1:2) lipid mixture mediated anti-fibroticdouble-stranded HJT-sRNA-m7 entry into MRC-5 cells.

As shown in FIG. 51, by boiling method and reverse evaporation method,No. 8 (PE):No. 12 (PC) (V:V=1:2) lipid mixture could effectively delivernucleic acid into cells to function.

Naive group: untreated MRC-5 cells, i.e., a blank control group;

TGF β G1 group: MRC-5 cells were stimulated with TGF β 1 protein (finalconcentration of 3 ng/mL), and the samples were collected after 72hours.

NC group: the mixture of lipid combination of No. 8 (PE):No. 12 (PC)(V:V=1:2) and double-stranded NC mimics was added to the MRC-5 cells andmixed well, and the final concentration of nucleic acid was 200 nM.After 24 hours, the cells were stimulated with TGFβ1 protein (finalconcentration of 3 ng/mL), and samples were collected 72 hours after thestimulation with TGFβ1.

M7 group: the mixture of lipid combination of No. 8 (PE):No. 12 (PC)(V:V=1:2) with double-stranded HJT-sRNA-m7 was added to the MRC-5 cellsand mixed, and the final concentration of nucleic acid was 200 nM. After24 hours, the cells were stimulated with TGFβ1 protein (finalconcentration of 3 ng/mL), and samples were collected after 72 hours.

2) No. 8 (PE):No. 12 (PC) (v:v=1:2) lipid mixture mediated siRNA entryinto A549 cells.

As shown in FIGS. 52 and 53, by the boiling method, lipid No. 8 (PE):No.12 (PC) (v:v=1:2) lipid mixture could effectively deliver nucleic acidinto cells to knockdown protein expression.

The naive group in FIG. 52: untreated cells, i.e., a blank controlgroup;

si-NC: the mixture of lipid combination of No. 8 (PE):No. 12 (PC)(v:v=1:2) and si-NC (synthesized by Guangzhou Ribobio Co., Ltd., unknownsequences) was added to A549 cells and mixed, and the finalconcentration was 400 nM; the cells were harvested after 48 hours, andlysed by RIPA strong lysis buffer to collect protein samples.

si-CPSF30: the mixture of lipid combination of No. 8 (PE):No. 12 (PC)(v:v=1:2) and si-CPSF30 was added to A549 cells and mixed, and the finalconcentration was 400 nM; the cells were harvested after 48 hours, andlysed by RIPA strong lysis buffer to collect protein samples.

si-LAMP1: the mixture of lipid combination of No. 8 (PE):No. 12 (PC)(v:v=1:2) and si-LAMP1 was added to A549 cells and mixed, the finalconcentration was 400 nM; the cells were harvested after 48 hours, andlysed by RIPA strong lysis buffer to collect protein samples.

si-LAMP2: the mixture of lipid combination of No. 8 (PE):No. 12 (PC)(v:v=1:2) and si-LAMP2 was added to A549 cells and mixed, and the finalconcentration was 400 nM; the cells were harvested after 48 hours, andlysed by RIPA strong lysis buffer to collect protein samples.

Free uptake group as shown in FIG. 53: the nucleic acid solution wasadded directly.

Lipo 2000 group: 2 μL Lipofectamine™ 2000 transfection reagent(Invitrogen, Thermo Fisher Scientific) and si-NF-κB solution werediluted in 100 μL opti-MEM medium, respectively, and the two were mixed,allowed to stay for 15 min, added to the cells and mixed, and the finalconcentration of nucleic acid solution was 400 nM; after 24 hours, thecells were stimulated with polyI:C (the concentration was 1 μg/mL), andthe protein samples were collected after 6 hours.

No. 8 (PE):No. 12 (PC) (1:2):No. 8 (PE):No. 12 (PC) (1:2) was mixed withthe si-NF-κB solution by heating method, then added to the cells, andthe final concentration of the nucleic acid solution was 400 nM; after24 hours, the cells were stimulated with polyI:C (the concentration was1 μg/mL), and the protein samples were collected after 6 hours.

See Table 2 for the types and sequences of the above nucleic acids.

3) No. 8 (PE):No. 12 (PC) (v:v=1:2) lipid mixture mediated siRNA entryinto THP-1 cells.

As shown in FIG. 54, by boiling method, No. 8 (PE):No. 12 (PC) (v:v=1:2)lipid mixture could effectively deliver nucleic acid into cells tofunction.

Naive group: untreated cells, i.e., a blank control group;

LPS group: no siRNA, but only LPS was added for stimulation, and thefinal concentration was 1 μg/mL. The RNA samples and cell supernatantswere harvested after 9 hours;

si-NC group: the mixture of lipid combination of No. 8 (PE):No. 12 (PC)(v:v=1:2) and si-NC was added to THP-1 cells and mixed, and the finalconcentration was 400 nM; LPS was added after 24 hours at a finalconcentration of 1 μg/mL for stimulation, and the TRIzol lysate of thecells were collected 9 hours after the stimulation, and the supernatantswere collected for ELISA detection.

si-TNFα group: the mixture of lipid combination of No. 8 (PE):No. 12(PC) (v:v=1:2) and si-TNFα was added to THP-1 cells and mixed, and thefinal concentration was 400 nM; LPS was added after 24 hours at a finalconcentration of 1 μg/mL for stimulation, the TRIzol lysate of the cellswere collected 9 hours after the stimulation, and the supernatants werecollected for ELISA detection.

2. No. 8 (PE):No. 12 (PC):No. 2 (DG) (v:v:v=2:4:3) lipid mixturemediated entry of nucleic acids into cells to function.

1) No. 8 (PE):No. 12 (PC):No. 2 (DG) (v:v:v=2:4:3) lipid mixturemediated anti-fibrotic HJT-sRNA-m7 entry into MRC-5 cells.

As shown in FIG. 55, by boiling method, No. 8 (PE):No. 12 (PC):No. 2(DG) (v:v:v=2:4:3) lipid mixture could effectively deliver anti-fibroticHJT-sRNA-m7 into MRC-5 cells to reduce fibronectin protein expression.

2) No. 8 (PE):No. 12 (PC):No. 2 (DG) (v:v:v=2:4:3) lipid mixturemediated XRN2 siRNA entry into A549 cells to inhibit gene expression.

As shown in FIG. 56, by boiling method, the addition of No. 2 (DG) tothe mixture of No. 8 (PE):No. 12(PC):No. 20 (DG), V:V:V=2:4:3 couldeffectively deliver nucleic acid into the cells to function.

Naive group: untreated A549 cells;

NC siRNA group: the mixture of lipid mixture of No. 8 (PE):No. 12(PC):No. 2 (DG) (v:v:v=2:4:3) and si-NC that was prepared by boilingmethod was added to the cells and mixed, and the final concentration ofthe nucleic acid was 400 nM;

XRN2 siRNA group: the mixture of lipid mixture of No. 8 (PE):No. 12(PC):No. 2 (DG) (v:v:v=2:4:3) and XRN2 siRNA that was prepared byboiling method was added to the cells and mixed, and the finalconcentration of the nucleic acid was 400 nM.

3. No. 8 (PE):No. 12 (PC):No. 4 (Cer) (v:v:v=1:2:1) lipid mixturemediated entry of nucleic acids into cells to function.

1) No. 8 (PE):No. 12 (PC):No. 4 (Cer) (v:v:v=1:2:1) lipid mixturemediated anti-fibrotic HJT-sRNA-m7 entry into MRC-5 cells (boilingmethod).

As shown in FIG. 57, by boiling method, the addition of No. 4 (Cer) tothe lipid mixture of No. 8 (PE), No. 12 (PC) (V:V=1:2), v:v:v=1:2:1,could effectively deliver anti-fibrotic HJT-sRNA-m7 into MRC-5 cells toreduce fibronectin protein expression.

Naive group: untreated cells;

TGF-β1 group: TGF-β1 protein was added at a final concentration of 3ng/mL for stimulation, and the samples were collected after 72 hours.

NC group: lipid combination of No. 38 (PE):No. 37 (LPC):No. 32 (TG)(V:V:V=32:8:5) was used to deliver NC mimics. After 24 hours, TGF-β1protein (final concentration of 3 ng/mL) was added for stimulation, andthe samples were collected after 72 hours.

m7 group: the mixture of lipid combination of No. 8 (PE):No. 12 (PC):No.4 (Cer) (V:V:V=1:2:1) with double-stranded HJT-sRNA-m7 was added to theMRC-5 cells and mixed, and the final concentration of nucleic acid was400 nM. After 24 hours, TGF-β1 protein (final concentration of 3 ng/mL)was added for stimulation, and the samples were collected after 72hours.

2) No. 8 (PE):No. 12 (PC):No. 4 (Cer) (v:v:v=1:2:1) lipid mixturemediated NF-κB siRNA entry into A549 cells to inhibit gene expression(boiling method).

As shown in FIG. 58, the addition of No. 4 (Cer) to a lipid mixture ofNo. 8 (PE), No. 12 (PC) (V:V=1:2), v:v:v=1:2:1, could effectivelydeliver nucleic acids into cells to function.

Naive group: untreated cells;

si-NC group: the mixture of lipid mixture of No. 8 (PE):No. 12 (PC):No.4 (Cer) (v:v:v=1:2:1) and siNC was added to cells and mixed, and thefinal concentration of the nucleic acid was 400 nM;

si-NF-κB group: the mixture of lipid mixture of No. 8 (PE):No. 12(PC):No. 4 (Cer) (v:v:v=1:2:1) and NF-κB siRNA was added to cells andmixed, the final concentration of the nucleic acid was 400 nM;

4. No. 8 (PE):No. 12 (PC):No. PC (11) (v:v:v=1:2:1) lipid mixturemediated entry of nucleic acids into cells to function.

1) No. 8 (PE):No. 12 (PC):No. PC (11) (v:v:v=1:2:1) lipid mixturemediated XRN2 siRNA entry into A549 cells to inhibit gene expression.

As shown in FIG. 59, the addition of No. 11 (PC) to the mixture of No. 8(PE), No. 12 (PC) (V:V=1:2), V:V:V=1:2:1, could effectively delivernucleic acid into the cells to function.

Naive group: untreated cells;

siNC group: the mixture of lipid mixture of No. 8 (PE):No. 12 (PC):No.PC (11) (v:v:v=1:2:1) and si-NC was added to the cells and mixed, andthe final concentration of the nucleic acid was 400 nM;

si-XRN2 group: the mixture of lipid mixture of No. 8 (PE):No. 12(PC):No. PC (11) (v:v:v=1:2:1) and XRN2 siRNA was added to the cells andmixed, and the final concentration of the nucleic acid was 400 nM.

5. No. 8 (PE):No. 12 (PC):No. LPC (37) (v:v:v=1:2:1) lipid mixturemediated entry of nucleic acids into cells to function.

1) No. 8 (PE):No. 12 (PC):No. LPC (37) (v:v:v=1:2:1) lipid mixturemediated XRN2 siRNA entry into A549 cells to inhibit gene expression.

As shown in FIG. 60, based on the addition of No. 37 (LPC) to the lipidmixture of No. 8 (PE), No. 12 (PC) (V:V=1:2), V:V:V=1:2:1, couldeffectively deliver nucleic acid into the cells to function.

Naive group: untreated cells;

si-NC group: the mixture of lipid mixture of No. 8 (PE):No. 12 (PC):No.LPC (37) (v:v:v=1:2:1) and si-NC was added to the cells and mixed, andthe final concentration of the nucleic acid was 400 nM;

si-XRN2 group: the mixture of lipid mixture of No. 8 (PE):No. 12(PC):No. LPC (37) (v:v:v=1:2:1) and XRN2 siRNA was added to the cellsand mixed, and the final concentration of the nucleic acid was 400 nM.

6. No. 8 (PE):No. 12 (PC):No. MG (34) (v:v:v=2:3:1) lipid mixturemediated entry of nucleic acids into cells to function.

1) No. 8 (PE):No. 12 (PC):No. MG (34) (v:v:v=2:3:1) lipid mixturemediated

CPSF4 siRNA entry into A549 cells to inhibit gene expression.

Naive group: untreated cells;

siNC group: the mixture of lipid mixture of No. 8 (PE):No. 12 (PC):No.MG (34) (v:v:v=2:3:1) and siNC was added to the cells and mixed, and thefinal concentration of the nucleic acid was 400 nM;

si-CPSF4 group: the mixture of lipid mixture of No. 8 (PE):No. 12(PC):No. MG (34) (v:v:v=2:3:1) and CPSF4 siRNA was added to the cellsand mixed, and the final concentration of the nucleic acid was 400 nM.

As shown in FIG. 61, No. 8 (PE):No. 12 (PC):No. MG (34) (v:v:v=2:3:1)lipid mixture could effectively deliver nucleic acid into the cells tofunction.

7. No. 38 (PE):No. 37 (LPC):No. 32 (TG) (v:v:v=32:8:5) lipid mixturemediated entry of nucleic acids into cells to function.

1) No. 38 (PE):No. 37 (LPC):No. 32 (TG) (v:v:v=32:8:5) lipid mixturemediated anti-fibrotic HJT-sRNA-m7 entry into MRC-5 cells (boilingmethod).

As shown in FIG. 62, the m7 band was lighter compared to control. Theeffect of M7 was not sufficient to restore cells to unstimulated levels.

Naive group: untreated cells, i.e., a blank control group;

TGF-β1 group: cells were stimulated with TGF-β1 protein (finalconcentration of 3 ng/mL), and the samples were collected after 72hours.

NC group: lipid combination of No. 38 (PE):No. 37 (LPC):No. 32 (TG)(V:V:V=32:8:5) was used to deliver NC mimics. After 24 hours, the cellswere stimulated with TGF-β1 protein (final concentration of 3 ng/mL),and the samples were collected after 72 hours.

M7 group: the mixture of lipid combination of No. 38 (PE):No. 37(LPC):No. 32 (TG) (V:V:V=32:8:5) with double-stranded HJT-sRNA-m7 wasadded to the MRC-5 cells and mixed, and the final concentration ofnucleic acid was 400 nM. After 24 hours, the cells were stimulated withTGF-β1 protein (final concentration of 3 ng/mL), and the samples werecollected after 72 hours.

2) No. 38 (PE):No. 37 (LPC):No. 32 (TG) (V:V:V=32:8:5) lipid mixturemediated XRN2 siRNA entry into A549 cells to inhibit gene expression.

As shown in FIG. 63, No. 38 (PE):No. 37 (LPC):No. 32 (TG) (V:V:V=32:8:5)lipid mixture could effectively deliver nucleic acid entering the cellsto function.

si-NC group: the mixture of lipid mixture of No. 38 (PE):No. 37(LPC):No. 32 (TG) (V:V:V=32:8:5) and si-NC was added to the cells andmixed, and the final concentration of the nucleic acid was 400 nM;

si-XRN2 group: the mixture of the lipid mixture of No. 38 (PE):No. 37(LPC):No. 32 (TG) (V:V:V=32:8:5) and XRN2 siRNA was added to the cellsand mixed, and the final concentration of the nucleic acid was 400 nM.

8. No. 1 (DG):No. 8 (PE):No. 12 (PC):No. 4 (Cer):No. 31 (So):No. 29(FA):No. 16 (TG) (v:v:v:v:v:v:v=2:1:2:2:3:1:3) lipid mixture mediatedentry of nucleic acids into cells to function.

1) As shown in FIG. 64, No. 1 (DG):No. 8 (PE):No. 12 (PC):No. 4(Cer):No. 31 (So):No. 29 (FA):No. 16 (TG) (v:v:v:v:v:v:v=2:1:2:2:3:1:3)lipid mixture mediated anti-fibrotic HJT-sRNA-m7 entry into MRC-5 cells(boiling method).

Naive group: untreated cells, i.e., a blank control group;

TGF-β1 group: cells were stimulated with TGF-β1 protein (finalconcentration of 3 ng/mL), and the samples were collected after 72hours.

NC group: lipid combination of No. 1 (DG):No. 8 (PE):No. 12 (PC):No. 4(Cer):No. 31 (So):No. 29 (FA):No. 16 (TG) (v:v:v:v:v:v:v=2:1:2:2:3:1:3)was used to was used to deliver NC mimics. After 24 hours, TGF-β1protein (final concentration of 3 ng/mL) was added for stimulation, andthe samples were collected after 72 hours.

M7 group: the mixture of lipid combination of No. 1 (DG):No. 8 (PE):No.12 (PC):No. 4 (Cer):No. 31 (So):No. 29 (FA):No. 16 (TG)(v:v:v:v:v:v:v=2:1:2:2:3:1:3) with single-stranded HJT-sRNA-m7 was addedto the MRC-5 cells, and mixed, and the final concentration of nucleicacid was 400 nM. After 24 hours, TGF-β1 protein (final concentration of3 ng/mL) was added for stimulation, and the samples were collected after72 hours.

2) As shown in FIG. 65, No. 1 (DG):No. 8 (PE):No. 12 (PC):No. 4(Cer):No. 31 (So):No. 29 (FA):No. 16 (TG) (v:v:v:v:v:v:v=2:1:2:2:3:1:3)lipid mixture mediated XRN2 siRNA entry into A549 cells to inhibit geneexpression (boiling method).

No. 1 (DG):No. 8 (PE):No. 12 (PC):No. 4 (Cer):No. 31 (So):No. 29(FA):No. 16 (TG) (v:v:v:v:v:v:v=2:1:2:2:3:1:3) lipid mixture couldeffectively deliver nucleic acid entering the cells to function.

si-NC group: the mixture of lipid mixture of No. 1 (DG):No. 8 (PE):No.12 (PC):No. 4 (Cer):No. 31 (So):No. 29 (FA):No. 16 (TG)(v:v:v:v:v:v:v=2:1:2:2:3:1:3) and si-NC was added to the cells andmixed, and the final concentration of the nucleic acid was 400 nM;

si-XRN2 group: the mixture of lipid mixture of No. 1 (DG):No. 8 (PE):No.12 (PC):No. 4 (Cer):No. 31 (So):No. 29 (FA):No. 16 (TG)(v:v:v:v:v:v:v=2:1:2:2:3:1:3) and XRN2 siRNA was added to the cells andmixed, and the final concentration of the nucleic acid was 400 nM.

9. No. 8 (PE):No. 12 (PC):No. 31 (So):No. 29 (FA):No. 4 (Cer)(v:v:v:v:v=2:4:2:2:5) lipid mixture mediated entry of nucleic acids intocells to function.

1) As shown in FIG. 66, No. 8 (PE):No. 12 (PC):No. 31 (So):No. 29(FA):No. 4 (Cer) (v:v:v:v:v=2:4:2:2:5) lipid mixture mediatedanti-fibrotic HJT-sRNA, HJT-sRNA-3, HJT-sRNA-a2, HJT-sRNA-h3,HJT-sRNA-m7 entry into MRC-5 cells (boiling method).

Naive group: untreated cells, i.e., a blank control group;

TGF-β1 group: cells were stimulated with TGF-β1 protein (finalconcentration of 3 ng/mL), and the samples were collected after 72hours.

NC group: lipid combination of No. 8 (PE):No. 12 (PC):No. 31 (So):No. 29(FA):No. 4 (Cer) (v:v:v:v:v=2:4:2:2:5) was used to deliver NC mimics.After 24 hours, the cells were stimulated with TGF-β1 protein (finalconcentration of 3 ng/mL), and the samples were collected after 72hours.

M7 group: the mixture of lipid combination of No. 8 (PE):No. 12 (PC):No.31 (So):No. 29 (FA):No. 4 (Cer) (v:v:v:v:v=2:4:2:2:5) with HJT-sRNA-m7single-stranded was added to the MRC-5 cells, mixed, and the finalconcentration of nucleic acid was 400 nM. After 24 hours, the cells werestimulated with TGF-β1 protein (final concentration of 3 ng/mL), and thesamples were collected after 72 hours.

2) As shown in FIG. 67, No. 8 (PE):No. 12 (PC):No. 31 (So):No. 29(FA):No. 4 (Cer) (v:v:v:v:v=2:4:2:2:5) lipid mixture mediated XRN2 siRNAentry into A549 cells to inhibit gene expression (boiling method).

No. 8 (PE):No. 12 (PC):No. 31 (So):No. 29 (FA):No. 4 (Cer)(v:v:v:v:v=2:4:2:2:5) lipid mixture could effectively deliver nucleicacid into the cells to function.

si-NC group: the mixture of lipid mixture of No. 8 (PE):No. 12 (PC):No.31 (So):No. 29 (FA):No. 4 (Cer) (v:v:v:v:v=2:4:2:2:5) and si-NC wasadded to the cells and mixed, and the final concentration of the nucleicacid was 400 nM;

si-XRN2 group: the mixture of lipid mixture of No. 8 (PE):No. 12(PC):No. 31 (So):No. 29 (FA):No. 4 (Cer) (v:v:v:v:v=2:4:2:2:5) and XRN2siRNA was added to the cells and mixed, and the final concentration ofthe nucleic acid was 400 nM.

10. No. 38 (PE):No. 37 (LPC) (v:v=4:1) lipid mixture mediated entry ofnucleic acids into cells to function.

1) As shown in FIG. 68, No. 38 (PE):No. 37 (LPC) (v:v=4:1) lipid mixturemediated anti-fibrotic HJT-sRNA, HJT-sRNA-3, HJT-sRNA-a2, HJT-sRNA-h3,HJT-sRNA-m7 entry into MRC-5 cells (boiling method).

Naive group: untreated cells, i.e., a blank control group;

TGF-β1 group: cells were stimulated with TGF-β1 protein (finalconcentration of 3 ng/mL), and the samples were collected after 72hours.

NC group: lipid combination of No. 38 (PE):No. 37 (LPC) (v:v=4:1) wasused to was used to deliver NC mimics. After 24 hours, the cells werestimulated with TGF-β1 protein (final concentration of 3 ng/mL), and thesamples were collected after 72 hours.

M7 group: the mixture of lipid combination of No. 38 (PE):No. 37 (LPC)(v:v=4:1) with HJT-sRNA-3, HJT-sRNA-a2, HJT-sRNA-h3, HJT-sRNA-m7 wasadded to the MRC-5 cells, and mixed, and the final concentration ofnucleic acid was 400 nM. After 24 hours, the cells were stimulated withTGF-β1 protein (final concentration of 3 ng/mL), and the samples werecollected after 72 hours.

2) As shown in FIG. 69, No. 38 (PE):No. 37 (LPC) (v:v=4:1) lipid mixturemediated XRN2 siRNA entry into A549 cells to inhibit gene expression(boiling method).

No. 38 (PE):No. 37 (LPC) (v:v=4:1) lipid mixture could effectivelydeliver nucleic acid entering the cells to function.

si-NC group: the mixture of lipid mixture of No. 38 (PE):No. 37 (LPC)(v:v=4:1) and si-NC was added to the cells and mixed, and the finalconcentration of the nucleic acid was 400 nM;

si-XRN2 group: the mixture of lipid mixture of No. 38 (PE):No. 37 (LPC)(v:v=4:1) and XRN2 siRNA was added to the cells and mixed, and the finalconcentration of the nucleic acid was 400 nM.

11. No. 38 (PE):No. 12 (PC):No. 2 (DG) (v:v:v=4:1:3) lipid mixturemediated entry of nucleic acids into cells to function.

As shown in FIG. 70, No. 38 (PE):No. 12 (PC):No. 2 (DG) (v:v:v=4:1:3)lipid mixture mediated XRN2 siRNA entry into A549 cells to inhibit geneexpression.

The lipid mixture of No. 38 (PE), in place of No. 8 (PE), with No. 12(PC), No. 2 (DG) (v:v:v=4:1:3) could effectively deliver nucleic acidentering the cells to function.

si-NC group: the mixture of lipid mixture of No. 38 (PE):No. 12 (PC):No.2 (DG) (v:v:v=4:1:3) and si-NC was added to the cells and mixed, and thefinal concentration of the nucleic acid was 400 nM;

si-XRN2 group: the mixture of lipid mixture of No. 38 (PE):No. 12(PC):No. 2 (DG) (v:v:v=4:1:3) and XRN2 siRNA was added to the cells andmixed, and the final concentration of the nucleic acid was 400 nM.

12. No. 38 (PE):No. 37 (LPC):No. 12 (PC) (v:v:v=4:1:1) lipid mixturemediated entry of nucleic acids into cells to function.

As shown in FIG. 71, No. 38 (PE):No. 37 (LPC):No. 12 (PC) (v:v:v=4:1:1)lipid mixture mediated XRN2 siRNA entry into A549 cells to inhibit geneexpression (reverse evaporation method).

The addition of No. 12 (PC) (v:v:v=4:1:1) to the lipid mixture of No. 38(PE):No. 37 (LPC) (v:v=4:1), could effectively deliver nucleic acid intocells to inhibit gene expression.

si-NC group: the mixture of lipid mixture of No. 38 (PE):No. 37(LPC):No. 12 (PC) (v:v:v=4:1:1) and si-NC was added to the cells andmixed, and the final concentration of the nucleic acid was 400 nM;

si-RNA group: the mixture of lipid mixture of No. 38 (PE):No. 37(LPC):No. 12 (PC) (v:v:v=4:1:1) and XRN2 siRNA, β-actin siRNA, Ssu 72siRNA or CPSF4 siRNA was added to the cells and mixed, and the finalconcentration of the nucleic acid was 400 nM.

13. No. 4 (Cer):No. 12 (PC):No. 38 (PE):No. 37 (LPC) (v:v:v:v=5:2:8:3)lipid mixture mediated entry of nucleic acids into cells to function.

1) As shown in FIG. 72, the addition of No. 4 (Cer) to the lipid mixtureof No. 38 (PE), No. 37 (LPC), No. 12 (PC) led to the lipid mixture ofNo. 4 (Cer):No. 12 (PC):No. 38 (PE):No. 37 (LPC) (v:v:v:v=5:2:8:3),which mediated anti-fibrotic HJT-sRNA, HJT-sRNA-3, HJT-sRNA-a2,HJT-sRNA-h3, HJT-sRNA-m7 double-stranded RNA entry into MRC-5 cells toreduce fibronectin expression levels (boiling method).

Naive group: untreated cells, i.e., a blank control group;

TGF-β1 group: cells were stimulated with TGF-β1 protein (finalconcentration of 3 ng/mL), and the samples were collected after 72hours.

NC group: lipid combination of No. 4 (Cer):No. 12 (PC):No. 38 (PE):No.37 (LPC) (v:v:v:v=5:2:8:3) was used to deliver NC mimics, after 24hours, TGF-β1 protein (final concentration of 3 ng/mL) was added forstimulation, and the samples were collected after 72 hours.

HJT-3 & a2 & h3 group: the mixture of lipid mixture of No. 4 (Cer):No.12 (PC):No. 38 (PE):No. 37 (LPC) (v:v:v:v=5:2:8:3) with HJT-sRNA-3,HJT-sRNA-a2, HJT-sRNA-h3 and HJT-sRNA-m7 double-strand, was added to thecells, and mixed and the final concentration of nucleic acid was 400 nM.

m7 group: the mixture of lipid combination of No. 4 (Cer):No. 12(PC):No. 38 (PE):No. 37 (LPC) (v:v:v:v=5:2:8:3) with HJT-sRNA-m7 wasadded to the cells, and mixed, and the final concentration of nucleicacid was 400 nM.

2) As shown in FIG. 73, No. 4 (Cer):No. 12 (PC):No. 38 (PE):No. 37 (LPC)(v:v:v:v=5:2:8:3) lipid mixture mediated XRN2 siRNA entry into cells toinhibit gene expression.

si-NC group: the mixture of lipid mixture of No. 4 (Cer):No. 12 (PC):No.38 (PE):No. 37 (LPC) (v:v:v:v=5:2:8:3) and si-NC was added to the cellsand mixed, and the final concentration of the nucleic acid was 400 nM;

si-XRN2 group: the mixture of lipid mixture of No. 4 (Cer):No. 12(PC):No. 38 (PE):No. 37 (LPC) (v:v:v:v=5:2:8:3) and XRN2 siRNA was addedto the cells and mixed, and the final concentration of the nucleic acidwas 400 nM.

14. No. 38 (PE):No. 2 (DG):No. 31 (So) (v:v:v=4:2:3) lipid mixturemediated entry of nucleic acids into cells to function.

1) As shown in FIG. 74, No. 38 (PE):No. 2 (DG):No. 31 (So) (v:v:v=4:2:3)lipid mixture mediated anti-fibrotic HJT-sRNA, HJT-sRNA-3, HJT-sRNA-a2,HJT-sRNA-h3, HJT-sRNA-m7 double-stranded RNA entry into MRC-5 cells toreduce fibronectin expression levels (boiling method).

Naive group: untreated cells, i.e., a blank control group;

TGF-β1 group: cells were stimulated with TGF-β1 protein (finalconcentration of 3 ng/mL), and the samples were collected after 72hours.

NC group: lipid combination of No. 38 (PE):No. 2 (DG):No. 31 (So)(v:v:v=4:2:3) was used to deliver NC mimics. After 24 hours, TGF-β1protein (final concentration of 3 ng/mL) was added for stimulation, andthe samples were collected after 72 hours.

HJT-3 & a2 & h3 group: the mixture of lipid mixture of No. 38 (PE):No. 2(DG):No. 31 (So) (v:v:v=4:2:3) with HJT-sRNA-3, HJT-sRNA-a2 andHJT-sRNA-h3, was added to the cells, and mixed and the finalconcentration of nucleic acid was 400 nM.

M7 group: the mixture of lipid combination of No. 38 (PE):No. 37 (LPC)(v:v=4:1) with HJT-sRNA-m7 was added to the cells, and mixed, and thefinal concentration of nucleic acid was 400 nM.

2) As shown in FIG. 75, No. 38 (PE):No. 2 (DG):No. 31 (So) (v:v:v=4:2:3)lipid mixture mediated XRN2 siRNA entry into A549 cells to inhibit geneexpression (boiling method).

No. 38 (PE):No. 2 (DG):No. 31 (So) (v:v:v=4:2:3) lipid mixtureeffectively delivered XRN2 siRNA into A549 cells to function.

si-NC group: the mixture of lipid mixture of No. 38 (PE):No. 2 (DG):No.31 (So) (v:v:v=4:2:3) and si-NC was added to the cells and mixed, andthe final concentration of the nucleic acid was 400 nM;

si-XRN2 group: the mixture of lipid mixture of No. 38 (PE):No. 2(DG):No. 31 (So) (v:v:v=4:2:3) and XRN2 siRNA was added to the cells andmixed, and the final concentration of the nucleic acid was 400 nM.

Example 5: Validation of the Effects of Lipid No. 41 and its Composition

I. Single Lipids Delivered Nucleic Acids (Double-Stranded RNA andSingle-Stranded RNA) into Cells by Different Preparation Methods(Reverse Evaporation and Boiling Method)

Lipid No. 41. Sphinganine (d22:0)

1. Quantitative Real-Time PCR (Real-Time PCR) Detection of theEfficiency of Nucleic Acid Delivery by Lipid.

As shown in FIG. 76, lipid No. 41 prepared by different methods (boilingor reverse evaporation method) delivered HJT-sRNA-m7 double-stranded RNAinto A549 cells.

For A549 cells, in the case of the boiling method, the delivery effectof lipid No. 41 was about twice that of RNAiMAX, and in the case of thereverse evaporation method, the delivery effect of lipid No. 41 was alsosignificantly higher than that of RNAiMAX.

As shown in FIG. 77, lipid No. 41 prepared by different methods (boilingor reverse evaporation method) delivered HJT-sRNA-m7 double-stranded RNAinto MRC-5 cells.

For MRC-5 cells, in the case of the boiling method, lipid No. 41delivered double-stranded RNA into MRC-5 cells, and in the case of thereverse evaporation method, the delivery effect of lipid No. 41 wassignificantly higher than that of RNAiMAX.

As shown in FIG. 78, lipid No. 41 delivered HJT-sRNA-m7 single-strandedRNA into A549 and MRC-5 cells by the boiling method.

2. Digital PCR (ddPCR) Detection of the Efficiency of Nucleic AcidDelivery by Lipid

2.1 Experimental materials: A549 cells were purchased from the CellCenter of the Institute of Basic Medical Sciences, Chinese Academy ofMedical Sciences, TRIzol lysis buffer was purchased from Sigma, Highcapacity cRNA Reverse Transcription Kit was purchased from ABI, USA, andthe digital PCR related reagents were purchased from Bio-Rad USA.

2.2 Experimental method: the total cellular RNA was collected andextracted by TRIzol lysis buffer according to the above methods, andreverse transcribed to cDNA using High capacity cRNA ReverseTranscription Kit, and the cDNA from different groups was subject todigital PCR reaction. Refering to the QX200 Droplet Reader andQuantaSoft Software manual for the protocols, the results were analyzedusing QuantaSoft software. The groups were as follows: (1) naive group:A549 cells without treatment; (2) free uptake group: the cells weredirectly incubated with HJT-sRNA-m7 dsRNA for 6 hours; (3) RNAiMAXgroup: A549 cells were transfected with the HJT-sRNA-m7 dsRNA byRNAiMAX, and the samples were collected for detection after 6 hours; (4)No. 41 group: lipid No. 41 prepared by different methods (boiling methodor reverse evaporation method) delivered double-stranded RNA into A549cells, and samples were collected for detection after 6 hours.

Experimental results and analysis: as shown in FIG. 79, by both theboiling method and reverse evaporation method, lipid No. 41 couldeffectively deliver HJT-sRNA-m7 dsRNA into A549 cells, and the boilingmethod had better effects than reverse evaporation method.

3. Flow Cytometry Detection of the Efficiency of Nucleic Acid Deliveryby Lipid

Experimental materials: A549 cells (purchased from the Cell Center ofthe Chinese Academy of Medical Sciences), FAM-sRNA (purchased fromRibobio Biotechnology Co., Ltd.), lipid No. 41, Accuri® C6 instrument(purchased from BD, USA).

Experimental methods: PGY-sRNA-6-FAM was dissolved in 100 μl water,mixed with 4 μl lipid, and prepared by boiling method. Then the mixturewas dropped into A549 cells, and after 6 hours of co-incubation, thesamples were collected for detection as follows: firstly wash threetimes with PBS, then digest with trypsin for 3 minutes and removetrypsin, wash with PBS again and then blow down the cells. The detectionwas performed using Accuri® C6 instrument.

Experimental results as shown in FIG. 80: lipid No. 41 had an efficiencyof 94.1% in delivering PGY-sRNA-6 single-stranded RNA, which was higherthan 69.4% of the positive control RNAiMAX. And lipid No. 41 had anefficiency of 96.7% in delivering

PGY-sRNA-6 double-stranded RNA, which was also higher than 94.9% of thepositive control RNAiMAX. Lipids 41 could efficiently deliversingle-stranded and double-stranded nucleic acids into A549 cells.

4. Observe of the Localization of the Nucleic Acid Delivered by Lipid inCells by Confocal Fluorescence Microscopy

Experimental materials: A549 cells (purchased from the Cell Center ofthe Chinese Academy of Medical Sciences), PGY-sRNA-6-Cy3 (purchased fromRibobio Biotechnology Co., Ltd.), lipid No. 41, Zeiss LSM780 (purchasedfrom Zeiss, Germany), Alexa Fluor® 488 phalloidin (purchased fromInvitrogen, USA), DAPI (purchased from Invitrogen, USA),paraformaldehyde (purchased from sigma, USA).

Experimental methods: PGY-sRNA-6-FAM was dissolved in 100 μl water,mixed with 4 μl lipid, and prepared by the boiling method. Then themixture was dropped into A549 cells, and after 6 hours of co-incubation,the samples were washed three times with PBS, fixed with 4%paraformaldehyde, washed three times with PBS, stained with Alexa Fluor®488 phalloidin for 30 min, washed 3 times with PBS, and stained withDAPI for 5 min, washed with PBS, and then sealed.

Experimental results as shown in FIG. 81: the entry of redPGY-sRNA-6-Cy3 could be obviously observed under the confocalmicroscopy. Lipid No. 41 could effectively deliver double-strandednucleic acid into A549 cells.

5. Western Blot Detection of the Efficiency of Nucleic Acid Delivery byLipid

As shown in FIG. 82, single lipid No. 41 mediated sRNAi entry intoMRC-5A549 cells to knockdown protein expression (by reverse evaporationmethod). At protein level, the protein knockdown effect mediated by thesingle lipid No. 41 was significantly higher than the inhibitory effectof HJT-sRNA-m7 mediated by RNAiMAX.

Naive group: untreated MRC-5A549 cells.

siNC group: the mixture of single lipid No. 41 and siNC was added to thecells and mixed, and the final concentration of the nucleic acid was 400nM;

siRNA group: the mixture of single lipid No. 41 and LAMP2, XPN2, Ssu72,CPSF4 or β-actin siRNA was added to the cells, mixed, and the finalconcentration of the nucleic acid was 400 nM;

Free uptake group: the test substance was directly added;

RNAiMAX group: 2 μl RNAiMAX transfection reagent and nucleic acidsolution were diluted with 100 μl opti-MEM medium, respectively, and thetwo were mixed, allowed to stay for 15 min, added to the cells, and thenmixed, and the final concentration of nucleic acid was 400 nM;

So (41) group (reverse evaporation method): the mixture of lipid No. 41and the nucleic acid was added to the cells and mixed, and the finalconcentration of the nucleic acid was 400 nM;

As shown in FIG. 83, single lipid No. 41 mediated anti-fibroticHJT-sRNA-m7 double-strand entry into MRC-5 cells (reverse evaporationmethod). At protein level, single lipid No. 41 mediated HJT-sRNA-m7inhibition was higher than RNAiMAX mediated HJT-sRNA-m7 inhibition.

TGF β 1 group: TGF-β 1 protein (final concentration was 3 ng/mL) wasadded for stimulation, and the samples were collected after 72 hours;

NC group: single lipid No. 41 delivered NC mimics. After 24 hours, thecells were stimulated with TGF-β1 protein (final concentration was 3ng/mL), and the samples were collected after 72 hours;

HJT-3 & a2 & H3 group: the mixture of single lipid No. 41 andHJT-sRNA-3, HJT-sRNA-a2 and HJT-sRNA-h3 were added to the cells andmixed, and the final concentration of the nucleic acid was 400 nM;

m7 group: the mixture of single lipid No. 41 and HJT-sRNA-m7 was addedto the cells and mixed, and the final concentration of the nucleic acidwas 400 nM;

6. Summary of In Vivo Results of Lipid No. 41

[Experimental Method]

6-8 weeks old mice, 22-24 g, were raised in SPF room of the AnimalCenter of the Institute of Basic Medical Sciences of Chinese Academy ofMedical Sciences. The mice were fasted for 12 hours before intragastricadministration. The mice were randomly divided into 3 groups: (1)control group, 400 μl DEPC-treated water, intragastric administration;(2) free uptake group, small RNA (PGY-sRNA-26, PGY-sRNA-32 andPGY-sRNA-23), each small RNA 1 nmol/animal, dissolved in 400 μlDEPC-treated water, intragastric administration; (3) lipid No. 41 group:a mixture of small RNA (PGY-sRNA-26 and PGY-sRNA-32) and lipid No. 41prepared by heating method was intragastrically administered, each smallRNA 1 nmol/animal, lipid No. 41 10 μl/animal, dissolved in 400 μlDEPC-treated water. All tissue and organ samples were collected after 6hours of intragastric administration. All small RNAs weresingle-stranded RNA modified by 3p-terminal 2-O-methylation.

[Experimental Results]

As shown in FIG. 108, lipid No. 41 could promote the entry of small RNAinto the blood, protecting it from degradation in the blood.

As shown in FIG. 109, lipid No. 41 could promote the entry of small RNAinto the stomach cells, protecting it from degradation in the stomach.

As shown in FIG. 110, lipid No. 41 could promote the entry of small RNAinto small intestinal cells, protecting it from degradation in the smallintestine.

As shown in FIG. 111, lipid No. 41 could promote the entry of small RNAinto the liver, protecting it from degradation in the liver.

7. Effect of lipid combination containing lipid No. 41 on nucleic aciddelivery 1) Effect of lipid combination 1 (No. 8+No. 41=6:1) and lipidcombination 2 (No. 38+No. 41=6:1) on nucleic acid delivery.

As shown in FIG. 84, lipid combination 1 (No. 8+No. 41=6:1) and lipidcombination 2 (No. 38+No. 41=6:1) mediated anti-fibrotic HJT-3 & a2 &H3, HJT-sRNA-m7 entry into MRC-5 cells (heating method), and mediated asignificant inhibitory effect of the HJT-sRNA-m7 at protein level.

TGF: TGF-β1 protein (final concentration was 3 ng/mL) was added forstimulation, and the samples were collected after 72 hours;

NC group: single lipid No. 41 was used to deliver NC mimics. After 24hours, TGF-β1 protein (final concentration was 3 ng/mL) was added forstimulation, and the samples were collected after 72 hours;

HJT-3 & a2 & H3 group: the mixture of the lipid mixture with HJT-sRNA-3,HJT-sRNA-a2 and HJT-sRNA-h3 was added to the cells and mixed, and thefinal concentration of the nucleic acid was 400 nM;

HJT-m7: the mixture of the lipid mixture and HJT-sRNA-m7 was added tothe cells and mixed, and the final concentration of the nucleic acid was400 nM;

2) Effects of lipid combination 3 (No. 39+No. 41=6:1) and lipidcombination 4 (No. 40+No. 41=6:1) on nucleic acid delivery.

As shown in FIG. 85, lipid combination 3 (No. 39+No. 41=6:1) and lipidcombination 4 (No. 40+No. 41=6:1) mediated anti-fibrotic HJT-3 & a2 &H3, HJT-sRNA-m7 entering into MRC-5 cells (heating method), and mediateda significant inhibitory effect of HJT-sRNA-m7 at protein the level.

TGF: TGF-β1 protein (final concentration was 3 ng/mL) was added forstimulation, and the samples were collected after 72 hours;

NC group: single lipid No. 41 was used to deliver NC mimics. After 24hours, the TGF-β1 protein (final concentration was 3 ng/mL) was addedfor stimulation, and the samples were collected after 72 hours;

HJT-3 & a2 & H3 group: the mixture of the lipid mixture with HJT-sRNA-3,HJT-sRNA-a2 and HJT-sRNA-H3 was added to the cells and mixed, and thefinal concentration of the nucleic acid was 400 nM;

HJT-m7: the mixture of the lipid mixture and HJT-sRNA-m7 was added tothe cells and mixed, and the final concentration of the nucleic acid was400 nM;

3) Effect of lipid combination 5 (No. 38+12+41+29=1:2:1:1) on nucleicacid delivery.

As shown in FIG. 86, lipid combination 5 (No. 38+12+41+29=1:2:1:1)mediated anti-fibrotic HJT-3 & a2 & H3 and HJT-sRNA-m7 entering intoMRC-5 cells (heating method), and mediated a significant inhibitoryeffect of HJT-sRNA-m7 at the protein level.

TGF: TGF-β1 protein (final concentration was 3 ng/mL) was added forstimulation, and samples were collected after 72 hours;

NC group: single lipid No. 41 was used to deliver NC mimics. After 24hours TGF-β1 protein (final concentration was 3 ng/mL) was added forstimulation, and the samples were collected after 72 hours;

HJT-3 & a2 & H3 group: the mixture of the lipid mixture with HJT-sRNA-3,HJT-sRNA-a2 and HJT-sRNA-H3 mixture was added to the cells and mixed,and the final concentration of the nucleic acid was 400 nM;

HJT-m7: a mixture of the lipid mixture and HJT-sRNA-m7 was added to thecells and mixed, and the final concentration of the nucleic acid was 400nM;

4) Effect of lipid combination 6 (No. 40 (PE)+No. 12 (PC)+No. 41(So)=2:4:3) on nucleic acid delivery.

As shown in FIG. 87, lipid combination 6 (No. 40 (PE)+No. 12 (PC)+No. 41(So)=2:4:3) mediated anti-fibrotic HJT-3 & a2 & H3, HJT-sRNA-m7 enteringinto MRC-5 cells (boiling and reverse evaporation method), and mediateda significant inhibitory effect of the HJT-3 & a2 & H3, HJT-sRNA-m7 atthe protein level.

TGF: TGF-β1 protein (final concentration was 3 ng/mL) was added forstimulation, and samples were collected after 72 hours;

3′-NC group: single lipid No. 41 was used to deliver NC mimics, andafter 24 hours TGF-β1 protein (final concentration was 3 ng/mL) wasadded for stimulation, and samples were collected after 72 hours;

3′-3 & a2 & H3 group: the mixture of lipid mixture with HJT-sRNA-3,HJT-sRNA-a2, HJT-sRNA-H3 was added to the cells and mixed, and the finalconcentration of the nucleic acid was 400 nM;

3′-m7: a mixture of lipid mixture and HJT-sRNA-m7 was added to thecells, mixed, and the final concentration of the nucleic acid was 400nM;

Right Figure: lipid-RNA mixture was prepared by reverse evaporation.Lipid combination 6 (No. 40 (PE)+No. 12 (PC)+No. 41 (So)=2:4:3) couldeffectively deliver XRN2, Ssu72, CPSF4 siRNA into A549 Cells, whichsignificantly reduce expression levels at the protein level.

siNC: the mixture of lipid mixture and siNC was added to the cells andmixed, and the final concentration of the nucleic acid was 400 nM;

siRNA: the mixture of lipid mixture and XRN2, Ssu72, CPSF4 siRNA wereadded to the cells, mixed, and the final concentration of the nucleicacid was 400 nM;

5) Effect of lipid combination 7 (No. 12 (PC)+No. 41 (So)=6:1) and lipidcombination 8 (No. 12 (PC)+No. 41 (So)=6:1) on nucleic acid delivery.

As shown in FIG. 88, by the reverse evaporation method, lipidcombination 7 (No. 12 (PC)+No. 41 (So)=6:1) and lipid combination 8 (No.12 (PC)+No. 41 (So)=6:1) could effectively deliver Ssu72, CPSF4 siRNAinto A549 Cells, which significantly reduced the expression levels atthe protein level.

siNC: the mixture of lipid mixture and siNC was added to the cells andmixed, and the final concentration of the nucleic acid was 400 nM;

siRNA: the mixture of lipid mixture and XRN2, Ssu72, CPSF4 siRNA wasadded to the cells, mixed, and the final concentration of the nucleicacid was 400 nM;

6) Effect of lipid combination 9 (No. 12 (PC)+No. 41 (So)=6:1) and lipidcombination 10 (No. 40 (PE)+No. 12 (PC)+No. 41 (So)=2:2:2) on nucleicacid delivery.

As shown in FIG. 89, by the reverse evaporation method, lipidcombination 9 (No. 12 (PC)+No. 41 (So)=6:1) and lipid combination 10(No. 40 (PE)+No. 12 (PC)+No. 41 (So)=2:2:2) could effectively deliverXRN2, Ssu72, CPSF4 siRNA into A549 Cells, which significantly reducedthe expression levels at the protein level.

siNC: the mixture of lipid mixture and siNC was added to the cells andmixed, and the final concentration of the nucleic acid was 400 nM;

siRNA: the mixture of lipid mixture and XRN2, Ssu72, CPSF4 siRNA wasadded to the cells, mixed, and the final concentration of the nucleicacid was 400 nM;

7) Effect of lipid combination 11 (No. 4 (Cer)+No. 12 (PC)+No. 41(So)=1:1:1) on nucleic acid delivery.

As shown in FIG. 90, by the reverse evaporation method, lipidcombination 11 (No. 4 (Cer)+No. 12 (PC)+No. 41 (So)=1:1:1) couldeffectively deliver Ssu72 siRNA into A549 Cells, which significantlyreduced the expression levels at protein level.

siNC: the mixture of lipid mixture and siNC was added to the cells andmixed, and the final concentration of the nucleic acid was 400 nM;

siSsu72: the mixture of lipid mixture and Ssu72 siRNA was added to thecells, mixed, and the final concentration of the nucleic acid was 400nM;

Example 6: Validation of the Effect of Lipid No. 38 and its Combination

Lipid No. 38 PE (16:0/16:1)

1. Quantitative Real-Time PCR (Real-Time PCR) Detection of theEfficiency of the Nucleic Acid Delivery by Lipid

(1) Lipid No. 38 by boiling method delivered double-stranded RNA intoA549 and MRC-5 cells.

As shown in FIG. 91, lipid No. 38 by heating method delivereddouble-stranded RNA into A549 and MRC-5 cells. For MRC-5 cells, in thecase of the heating method, the delivery effect of lipid No. 38 ondouble-stranded RNA was about twice that of RNAiMAX.

1) Naive group: untreated A549 cells;

2) Free uptake group: HJT-sRNA-m7 dsRNA was directly incubated withcells for 12 hours; the final concentration of nucleic acid was 100 nM;

3) RNAiMAX group: 2 μL RNAiMAX transfection reagent and double-strandedHJT-sRNA-m7 solution were diluted in 100 μL opti-MEM mediumrespectively, and then the two were mixed, allowed to stay for 15 min,added into the cells, and then mixed. The final concentration ofHJT-sRNA-m7 double-strand was 100 nM;

4) Treatment group of lipid and nucleic acid: a mixture of 2.5 μL singlelipid No. 38 and HJT-sRNA-m7 double-stranded nucleic acid solution wasprepared by boiling method or reverse evaporation method, and then addedto A549 cells. The final concentration of RNA was 100 nM. After 12hours, the sample was collected to detect the amount of entry.

(2) Lipid No. 38 by boiling method delivered HJT-sRNA-m7 single-strandedRNA into A549 and MRC-5 cells.

As shown in FIG. 92, lipid No. 38 by heating method deliveredHJT-sRNA-m7 single-stranded RNA into A549 and MRC-5 cells, where theefficiency of delivery was much higher than that of RNAiMAX.

1) Naive group: untreated A549 cells;

2) Free uptake group: HJT-sRNA-m7 single stranded RNA was directlyincubated with cells for 12 hours; the final concentration of nucleicacid was 100 nM;

3) RNAiMAX group: 2 μL RNAiMAX transfection reagent and single-strandedHJT-sRNA-m7 solution were diluted in 100 μL opti-MEM mediumrespectively, and then the two were mixed, allowed to stay for 15 min,added into the cells, and then mixed, and the final concentration ofsingle-stranded HJT-sRNA-m7 was 100 nM;

4) Treatment group of lipid and nucleic acid: a mixture of 2.5 μL singlelipid No. 64 and HJT-sRNA-m7 double-stranded nucleic acid solution wasprepared by boiling method or reverse evaporation method, and added toA549 cells, the final concentration of RNA was 100 nM. After 12 hours,the sample was collected to detect the amount of entry.

2. Digital PCR (ddPCR) Detection of the Efficiency of Nucleic AcidDelivery by Lipid

2.1 Experimental materials: A549 cells were purchased from the CellCenter of the Institute of Basic Medical Sciences, Chinese Academy ofMedical Sciences, TRIzol lysis buffer was purchased from Sigma, Highcapacity cRNA Reverse Transcription Kit was purchased from ABI, USA, anddigital PCR related reagents were purchased from Bio-Rad.

2.2 Experimental method: total RNA was collected and extracted by TRIzollysis buffer according to the above method, and reverse transcribed tocDNA using High capacity cRNA Reverse Transcription Kit, and the cDNAfrom different groups was subjected to digital PCR reaction. Refer tothe QX200 Droplet Reader and QuantaSoft Software manual for theprotocols; the results were analyzed using QuantaSoft software.

(1) Naive group: A549 cells without any treatment;

(2) Free uptake group: the cells were directly co-incubated withHJT-sRNA-m7 dsRNA for 6 hours;

(3) RNAiMAX group: the HJT-sRNA-m7 dsRNA was transfected into A549 cellsby RNAiMAX, and the samples were collected for detection after 6 hours;

(4) No. 38 group: lipid No. 38 delivered double-stranded RNA into A549cells by different preparation methods (boiling or evaporation method),and the samples were collected for detection after 6 hours;

Experimental results and analysis: As shown in FIG. 93, in the boilingor reverse evaporation method, lipid No. 38 could effectively deliverHJT-sRNA-m7 dsRNA into A549 cells.

3. Flow Cytometry Detection of the Efficiency of Nucleic Acid Deliveryby Lipid

Experimental materials: A549 cells (purchased from the Cell Center ofthe Chinese Academy of Medical Sciences), FAM-sRNA (purchased fromRibobio Biotechnology Co., Ltd.), lipid No. 38, Accuri® C6 instrument(purchased from BD, USA).

Experimental Method: PGY-sRNA-6-FAM was dissolved in 100 μl water, andmixed with 4 μl lipid, and prepared into lipid-sRNA mixture by boilingmethod. Then, the mixture was dropped into A549 cells, and after 6 hoursof co-incubation, the samples were collected and washed three times withPBS, then digested with trypsin into single cells, washed withre-suspended with PBS and then blown down for Accuri® C6 instrumentdetection.

Experimental results (shown in FIG. 94): lipid No. 38 deliveredPGY-sRNA-6 single-stranded RNA at an efficiency of 72.5%, which wasclose to that of the positive control RNAiMAX.

4. Confocal Fluorescence Microscopy to Observe the Location of theNucleic Acid Delivered by Lipids in Cells

Experimental materials: A549 cells (purchased from the Cell Center ofthe Chinese Academy of Medical Sciences), PGY-sRNA-6-Cy3 (purchased fromRibobio Biotechnology Co., Ltd.), lipid No. 38, Zeiss LSM780 (purchasedfrom Zeiss, Germany), Alexa Fluor® 488 phalloidin (purchased fromInvitrogen, USA), DAPI (purchased from Invitrogen, USA),paraformaldehyde (purchased from sigma, USA).

Experimental method: PGY-sRNA-6-FAM was dissolved in 100 μl water, andmixed with 4 μl lipid, and prepared by boiling method. Then, the mixturewas dropped into A549 cells, and after 6 hours of co-incubation, thesamples were washed three times with PBS, fixed with 4%paraformaldehyde, washed three times with PBS, stained with Alexa Fluor®488 phalloidin for 30 min, washed 3 times with PBS, and stained withDAPI for 5 min, PBS washed, and then sealed.

Experimental results (shown in FIG. 95): the entry of red PGY-sRNA-6-Cy3could be obviously observed under the confocal microscopy. Lipid No.38-sRNA mixture prepared by boiling method could effectively deliverdouble-stranded nucleic acid into A549 cells.

Example 7: Validation of the Effect of Lipid No. 64 and its Composition

Lipid No. 64 PE (15:0/24:1 (15Z))

1. Quantitative Real-Time PCR (Real-Time PCR) Detection of theEfficiency of the Nucleic Acid Delivery by Lipid

(1) Lipid No. 64 prepared by different methods (boiling or reverseevaporation method) delivered HJT-sRNA-m7 double-stranded RNA into A549cells.

As shown in FIG. 96, lipid No. 64 delivered HJT-sRNA-m7 double-strandedRNA into A549 cells by different preparation methods (boiling or reverseevaporation method). For A549 cells, in the case of the boiling method,the delivery effect of lipid No. 64 was about 3 times that of RNAiMAX.

1) Naive group: untreated A549 cells;

2) Free uptake group: HJT-sRNA-m7 dsRNA was directly incubated withcells for 12 hours; the final concentration of nucleic acid was 100 nM;

3) RNAiMAX group: 2 μL RNAiMAX transfection reagent and double-strandedHJT-sRNA-m7 solution were diluted in 100 μL opti-MEM mediumrespectively, mixed, and allowed to stay for 15 min, added into thecells and mixed, and the final concentration of HJT-sRNA-m7double-strand was 100 nM;

4) Treatment group of lipid and nucleic acid: a mixture of 2.5 μL singlelipid No. 64 and HJT-sRNA-m7 double-stranded nucleic acid solution wasprepared by boiling method or reverse evaporation method and added toA549 cells, the final concentration of RNA was 100 nM. After 12 hours,the sample was collected to detect the amount of entry.

2. Flow Cytometry Detection of the Efficiency of Nucleic Acid Deliveryby Lipid

Experimental materials: A549 cells (purchased from the Cell Center ofthe Chinese Academy of Medical Sciences), FAM-sRNA (purchased fromRibobio Biotechnology Co., Ltd.), lipid No. 64, Accuri® C6 instrument(purchased from BD, USA).

Experimental Method: FAM-sRNA was dissolved in 100 μl water, and mixedwith 4 μl lipid, prepared by boiling method. Then, the lipid-sRNAmixture was dropped into A549 cells, and after 6 hours of co-incubation,the samples were collected and washed three times with PBS, thendigested into single cells with trypsin, re-suspended with PBS and thenblown down for Accuri® C6 instrument detection.

Experimental results (shown in FIG. 97): lipid No. 64 deliveredPGY-sRNA-6 single-stranded RNA with an efficiency of about a half (½) ofefficiency the positive control RNAiMAX.

3. Confocal Fluorescence Microscopy to Observe the Location of theNucleic Acid Delivered by Lipids in Cells

Experimental materials: A549 cells (purchased from the Cell Center ofthe Chinese Academy of Medical Sciences), PGY-sRNA-6-Cy3 (purchased fromRibobio Biotechnology Co., Ltd.), lipid No. 64, Zeiss LSM780 (purchasedfrom Zeiss, Germany), Alexa Fluor® 488 phalloidin (purchased fromInvitrogen, USA), DAPI (purchased from Invitrogen, USA),paraformaldehyde (purchased from sigma, USA).

Experimental method: PGY-sRNA-6-FAM was dissolved in 100 μl water, andmixed with 4 μl lipid, and prepared by boiling method. Then, the mixturewas dropped into A549 cells, and after 6 hours of co-incubation, thesamples were washed three times with PBS, fixed with 4%paraformaldehyde, washed three times with PBS, stained with Alexa Fluor®488 phalloidin for 30 min, washed 3 times with PBS, and stained withDAPI for 5 min, PBS washed, and then sealed.

Experimental results (shown in FIG. 98): the entry of red PGY-sRNA-6-Cy3could be obviously observed under the confocal microscopy. Lipid No. 64could effectively deliver single-stranded RNA into A549 cells.

Example 8: Validation of the Effect of Lipid No. 40 and its Composition

Lipid No. 40 PE (16:0/22:1)

1. Quantitative Real-Time PCR (Real-Time PCR) Detection of theEfficiency of the Nucleic Acid Delivery by Lipid

(1) Lipid No. 40 prepared by different methods (boiling or reverseevaporation method) delivered double-stranded RNA into A549 cells.

As shown in FIG. 99, lipid No. 40 by prepared by different methods(boiling or reverse evaporation method) delivered double-stranded RNAinto A549 cells. For A549 cells, in the case of the reverse evaporationmethod, delivery effect of lipid No. 40 was about a half (½) of that ofRNAiMAX.

1) Naive group: untreated A549 cells;

2) Free uptake group: HJT-sRNA-m7 dsRNA was directly incubated withcells for 12 hours; the final concentration of nucleic acid was 100 nM;

3) RNAiMAX group: 2 μL RNAiMAX transfection reagent and double-strandedHJT-sRNA-m7 solution were diluted in 100 μL opti-MEM mediumrespectively, and then the two were mixed, allowed to stay for 15 min,added into the cells, mixed, and the final concentration of HJT-sRNA-m7double-strand was 100 nM;

4) Treatment group of lipid and nucleic acid: a mixture of 2.5 μL singlelipid No. 40 and HJT-sRNA-m7 double-stranded nucleic acid solution wasprepared by boiling method or reverse evaporation method, and added toA549 cells. The final concentration of RNA was 100 nM. After 12 hours,the sample was collected to detect the amount of entry.

2. Digital PCR (ddPCR) Detection of the Efficiency of Nucleic AcidDelivery by Lipid

2.1 Experimental materials: A549 cells were purchased from the CellCenter of the Institute of Basic Medical Sciences, Chinese Academy ofMedical Sciences, TRIzol lysis buffer was purchased from Sigma, TaqMan™MicroRNA Reverse Transcription KitHigh was purchased from Thermo FisherTechnology, and digital PCR related reagents were purchased fromBio-Rad.

2.2 Experimental method: Total RNA was collected and extracted by TRIzollysis buffer according to the above method, and reverse transcribed tocDNA using TaqMan™ MicroRNA Reverse Transcription KitHigh, and the cDNAfrom different groups was subjected to digital PCR reaction. Refer tothe QX200 Droplet Reader and QuantaSoft Software manual for theprotocols; the results were analyzed using QuantaSoft software.

(1) Naive group: A549 cells without any treatment

(2) Free uptake group: the cells were directly co-incubated withHJT-sRNA-m7 dsRNA for 6 hours;

(3) RNAiMAX group: the HJT-sRNA-m7 dsRNA was transfected into A549 cellsby RNAiMAX, and the samples were collected for detection after 6 hours;

(4) No. 40 group: lipid No. 40 prepared by different methods (boiling orevaporation method) delivered double-stranded RNA into A549 cells, andthe samples were collected for detection after 6 hours;

Experimental results and analysis: As shown in FIG. 100, in the boilingor reverse evaporation method, lipid No. 40 could effectively deliverHJT-sRNA-m7 dsRNA into A549 cells.

3. Confocal Fluorescence Microscopy to Observe the Location of theNucleic Acid Delivered by Lipids in Cells

Experimental materials: A549 cells (purchased from the Cell Center ofthe Chinese Academy of Medical Sciences), PGY-sRNA-6-Cy3 (purchased fromRibobio Biotechnology Co., Ltd.), lipid No. 40, Zeiss LSM780 (purchasedfrom Zeiss, Germany), Alexa Fluor® 488 phalloidin (purchased fromInvitrogen, USA), DAPI (purchased from Invitrogen, USA),paraformaldehyde (purchased from sigma, USA).

Experimental method: PGY-sRNA-6-FAM was dissolved in 100 μl water, andmixed with 4 μl lipid, and prepared by boiling method. Then, the mixturewas dropped into A549 cells, and after 6 hours of co-incubation, thesamples were washed three times with PBS, fixed with 4%paraformaldehyde, washed three times with PBS, stained with Alexa Fluor®488 phalloidin for 30 min, washed 3 times with PBS, and stained withDAPI for 5 min, PBS washed, and then sealed.

Experimental results (shown in FIG. 101): the entry of redPGY-sRNA-6-Cy3 could be obviously observed under the confocalmicroscopy. Lipid No. 40 could effectively deliver single-stranded RNAinto A549 cells.

4. Western Blotting Detection of the Efficiency of Nucleic Acid Deliveryby Lipid

As shown in FIG. 102, phosphatidylethanolamine single lipid No. 40mediated anti-fibrotic double-stranded RNA HJT-sRNA-m7 entry into MRC-5cells to down-regulate fibronectin protein expression.

TGF: TGF-β1 protein (final concentration was 3 ng/mL) was added forstimulation, and the samples were collected after 72 hours;

3′-NC group: lipid mixture was used to deliver NC mimics and after 24hours, the cells were stimulated with TGF-β 1 protein (finalconcentration was 3 ng/mL), and the samples were collected after 72hours;

3′-m7 group: a mixture of lipid mixture and HJT-sRNA-m7 double-strandednucleic acid solution was added to the cells and mixed, and the finalconcentration of the nucleic acid was 400 nM;

Example 9: Validation of the Effect of Lipid No. 37

Lipid No. 37 LPC (18:3)

1. Quantitative Real-Time PCR (Real-Time PCR) Detection of theEfficiency of the Nucleic Acid Delivery by Lipid

(1) Lipid No. 37 delivered single-stranded RNA into A549 and MRC-5 cellsby boiling method.

As shown in FIG. 103, single-stranded RNA was delivered to A549 and MRCScells by boiling method.

1) Naive group: untreated A549 cells;

2) Free uptake group: HJT-sRNA-m7 dsRNA was directly incubated withcells for 3 hours; the final concentration of nucleic acid was 100 nM;

3) RNAiMAX group: 2 μL RNAiMAX transfection reagent and single-strandedHJT-sRNA-m7 solution were diluted in 100 μL opti-MEM mediumrespectively, mixed, and allowed to stay for 15 min, added into thecells, mixed, and the final concentration of HJT-sRNA-m7 single-strandwas 100 nM;

4) Treatment group of lipid and nucleic acid: a mixture of 2.5 μL singlelipid No. 37 and HJT-sRNA-m7 single-stranded nucleic acid solution wasprepared by boiling method or reverse evaporation method and added toA549 cells, the final concentration of RNA was 100 nM. After 3 hours,the sample was collected to detect the amount of entry.

Example 10: Validation of the Effect of Lipid No. 39

Lipid No. 39 PE (16:1-18:1)

1. Quantitative Real-Time PCR (Real-Time PCR) Detection of theEfficiency of the Nucleic Acid Delivery by Lipid

As shown in FIG. 104, Lipid No. 39 prepared by different methods(boiling or reverse evaporation method) delivered double-stranded RNAinto A549 cells

1) Naive group: untreated A549 cells;

2) Free uptake group: HJT-sRNA-m7 dsRNA was directly incubated withcells for 6 hours; the final concentration of nucleic acid was 100 nM;

3) RNAiMAX group: 2 μL RNAiMAX transfection reagent and double-strandedHJT-sRNA-m7 solution were diluted in 100 μL opti-MEM mediumrespectively, mixed, and allowed to stay for 15 min, added into thecells and mixed, and the final concentration of HJT-sRNA-m7double-strand was 100 nM;

4) Treatment group of lipid and nucleic acid: a mixture of 2.5 μL singlelipid No. 39 and HJT-sRNA-m7 double-stranded nucleic acid solution wasprepared by boiling method or reverse evaporation method and added toA549 cells, the final concentration of RNA was 100 nM. After 12 hours,the sample was collected to detect the amount of entry.

2. Digital PCR (ddPCR) Detection of the Efficiency of Nucleic AcidDelivery by Lipid

2.1 Experimental materials: A549 cells were purchased from the CellCenter of the Institute of Basic Medical Sciences, Chinese Academy ofMedical Sciences, TRIzol lysis buffer was purchased from Sigma, Highcapacity cRNA Reverse Transcription Kit was purchased from ABI, USA, anddigital PCR related reagents were purchased from Bio-Rad.

2.2 Experimental method: Total RNA was collected and extracted by TRIzollysis buffer according to the above method, and reversed to cDNA usingHigh capacity cRNA Reverse Transcription Kit, and the cDNA fromdifferent groups was subjected to digital PCR reaction. Refer to theQX200 Droplet Reader and QuantaSoft Software manual for the protocols;the results were analyzed using QuantaSoft software.

(1) Naive group: A549 cells without any treatment;

(2) Free uptake group: the cells were directly co-incubated withHJT-sRNA-m7 dsRNA for 6 hours; 12 hours;

(3) RNAiMAX group: the HJT-sRNA-m7 dsRNA was transfected into A549 cellsby RNAiMAX, and the samples were collected for detection after 6 hours,12 hours;

(4) No. 39 group: lipid No. 39 delivered double-stranded RNA into A549cells by reverse evaporation method, and the samples were collected fordetection after 6 hours, 12 hours;

As shown in FIG. 105, by the reverse evaporation method, lipid No. 39could effectively deliver HJT-sRNA-m7 dsRNA into A549 cells.

Example 11: Validation of the Effect of Lipid No. 60 and No. 62

Lipid No. 60 dMePE (16:1/16:1)

1. Quantitative Real-Time PCR (Real-Time PCR) Detection of theEfficiency of the Nucleic Acid Delivery by Lipid

As shown in FIG. 106, Lipid No. 60 prepared by different methods(boiling or reverse evaporation method) delivered double-stranded RNAinto A549 cells 7) Naive group: untreated A549 cells;

8) Free uptake group: HJT-sRNA-m7 dsRNA was directly incubated withcells for 6 hours; the final concentration of nucleic acid was 100 nM;

RNAiMAX group: 2 μL RNAiMAX transfection reagent and double-strandedHJT-sRNA-m7 solution were diluted in 100 μL opti-MEM mediumrespectively, and then the two were mixed, allowed to stay for 15 min,added into the cells, and then mixed, the the final concentration ofdouble-stranded HJT-sRNA-m7 was 100 nM;

4) Lipid and nucleic acid: a mixture of 2.5 μL single lipid No. 60 andHJT-sRNA-m7 double-stranded nucleic acid solution was prepared byboiling method or reverse evaporation method and added to cells, thefinal concentration of RNA was 100 nM.

After 12 hours, the sample was collected to detect the amount of entry.

Lipid No. 62 dMePE (16:1/18:1)

1. Quantitative Real-Time PCR (Real-Time PCR) Detection of theEfficiency of the Nucleic Acid Delivery by Lipid

As shown in FIG. 107, Lipid No. 62 prepared by different methods(boiling or reverse evaporation method) delivered double-stranded RNAinto A549 cells

1) Naive group: untreated A549 cells;

2) Free uptake group: HJT-sRNA-m7 dsRNA was directly incubated withcells for 6 hours; the final concentration of nucleic acid was 100 nM;

3) RNAiMAX group: 2 μL RNAiMAX transfection reagent and double-strandedHJT-sRNA-m7 solution were diluted in 100 μL opti-MEM mediumrespectively, and then the two were mixed, allowed to stay for 15 min,added into the cells, and then mixed, and the final concentration ofHJT-sRNA-m7 double-strand was 100 nM;

4) Treatment group of lipid and nucleic acid: a mixture of 2.5 μL singlelipid No. 62 and HJT-sRNA-m7 double-stranded nucleic acid solution wasprepared by boiling method or reverse evaporation method and added tocells, and the final concentration of RNA was 100 nM. After 12 hours,the sample was collected to detect the amount of entry.

In Vivo Delivery Experiment of Lipid Nucleic Acid Mixture

1. Experimental animals: C57 mice, male, approximately 6 weeks old.

2. Manufacture of lipid mixture: the preparation was conducted on thebasis of a dose of 10 μl lipid-1 nmol sRNA per mouse as follows:dissolve 1 nmol of each sRNA in 500 μl DEPC water, add 10 μl of thecorresponding lipid, pipette to mix thoroughly, and then naturally cooldown after water bath for 15 min at 90° C., and administer via gavage.

3. sRNA: PGY-sRNA-26, PGY-sRNA-32

4. Experimental groups:

1) Naive group: intragastric administration of 500 μl saline;

2) RNAiMAX treatment group: 10 μl RNAiMAX-1 nmol sRNA was mixedthoroughly and intragastrically administered to each mouse. This groupserved as a positive control group. RNAiMAX was purchased fromInvitrogen.

3) Free uptake group: sRNA solution (1 nmol/animal, 500 μL) was directlyadded, and the group served as a negative control;

4) Treatment group of lipid nucleic acid mixture: the lipid-sRNA mixtureprepared in the step 2 was intragastrically administrated.

5. Detection of the relative amount of entry:

1) Tissue sampling and extraction of RNA: 6 hours after gavage in mice,take 500 μl of blood from the eyeball, add 1.5 ml Trizol Reagent LS tothoroughly mix and lyse, add 3 ml Trizol Reagent (purchased fromInvitrogen) to the tissue samples and homogenize until complete lysis.Tissues sampled: live/stomach/small intestine.

2) Reverse transcription of sRNA to cDNA: Reverse Transcription Kit(High-Capacity cDNA Reverse Transcription Kits, Applied Biosystems, cat.no. 4368813), was used to reverse transcribe the total RNA to cDNA, andthe reverse system was as follows: template RNA (150 ng/μL) 10 μL, 10×RTbuffer, 2.0 μL, 25×dNTP Mix (100 mM) 0.8 μL, random primers 2.0 μL, theMultiScribe™ reverse Transcriptase 1.0 μL, RNase inhibitor 1.0 μL,nuclease-free H₂O 3.2 μL. After a brief centrifugation, the reaction wasloaded in a PCR reactor. The reaction conditions were as follows: (1)25° C., 10 min; (2) 37° C., 120 min; (3) 85° C., 5 min; (4) 4° C.,termination of the reaction. After the reaction, 20 μL RNase-free ddH₂Owas added to make up the final volume to 40 μL.

3) Quantitative PCR amplification reactions: the qPCR reaction systemhad a total volume of 10 μl, containing: 5 μl 2×SYBR Green Master Mix,0.5 μl forward primer (10 μM), 0.5 μL reverse primer, 1 μl cDNA byreverse transcription, 3 μl RNase-free dH₂O. LightCycler 480fluorescence quantitative PCR instrument was used, and the PCR reactionconditions were: 95° C., 5 min for pre-denaturation, followed by the PCRamplification cycle: (1) 95° C., 10 s; (2) 55° C., 10 s; (3) 72° C., 20s; a total of 40 cycles; 40° C. for 10 s in the end to cool down. Theforward primer and reverse primer of the amplification reaction wasdesigned and synthesized by Beijing Qing Ke New

Industrial Biotechnology Co., Ltd. (U6 F primer: GCGCGTCGTGAAGCGTTC (SEQID NO: 23), U6 R primer: GTGCAGGGTCCGAGGT (SEQ ID NO: 24)).

3) The relative expression amount was calculated by the 2-ΔCt method.

Example 12-1: Delivery of Single-Stranded Nucleic Acids by Single LipidNo. 41 In Vivo

1. Experimental animals: C57 mice, male, approximately 6 weeks old.

1) Naive group: intragastric administration of 500 μl saline;

2) RNAiMAX treatment group: 10 μl RNAiMAX-1 nmol sRNA was mixed andintragastrically administered to each mouse. This group served as apositive control group. RNAiMAX was purchased from Invitrogen.

3) Free uptake group: single-stranded sRNA mixture solution (1 nmoleach) was directly added (1 nmol each);

4) Treatment group of single lipid and nucleic acid mixture: a mixtureof 10 μL of single lipid (No. 41) with single-stranded sRNA mixturesolution (PGY-sRNA-23, PGY-sRNA-26 and PGY-sRNA-32, 1 nmol each) wastreated by heating method and then given to mice by intragastricadministration.

2. 12 hours after intragastric administration, the blood was taken fromthe eyeball, and various tissues (liver/stomach/small intestine) wassampled. TRIzol was used for full lysis and the RNA was extracted todetect the amount of entry.

Conclusion:

As shown in FIG. 108, single So (No. 41) could effectively deliver sRNAsingle-stranded nucleic acid into the mouse blood via oraladministration to protect sRNA from degradation, and the delivery effectwas better than POPC and Lipofectamine RNAiMAX.

As shown in FIG. 109, single So (No. 41) could effectively deliver sRNAsingle-stranded nucleic acid into the mouse stomach via oraladministration to protect sRNA from degradation.

As shown in FIG. 110, single So (No. 41) could effectively deliver sRNAsingle-stranded nucleic acid into the mouse small intestine via oraladministration to protect sRNA from degradation.

As shown in FIG. 111, single So (No. 41) could effectively deliver sRNAsingle-stranded nucleic acid into the mouse liver via oraladministration to protect sRNA from degradation.

Example 12-2: Delivery of Single-Stranded Nucleic Acids by Single LipidNo. 38 In Vivo

1. Experimental animals: C57 mice, male, approximately 6 weeks old.

1) Naive group: intragastric administration of 500 μl saline;

2) RNAiMAX treatment group: 10 μl RNAiMAX-1 nmol sRNA was mixed andintragastrically administered to each mouse. This group served as apositive control group. RNAiMAX was purchased from Invitrogen.

3) Free uptake group: single-stranded sRNA mixture solution (1 nmoleach) was directly added (each 1 nmol);

4) Treatment group of POPC and nucleic acid: a mixture of 10 μL POPC andsingle-stranded PGY-sRNA-32 sRNA (each 1 nmol) mixture solution that wastreated by heating method was given to mice by gavage.

5) Treatment group of single lipid and nucleic acid mixture: a mixtureof a 10 μL single lipid (No. 38) and single-stranded sRNA (PGY-sRNA-32)mixture solution (each 1 nmol) that was treated by heating method wasgiven to mice by gavage.

2. 12 hours after gavage, the blood was taken from the eyeball and lysedby TRIzol to extract RNA for the detection of the amount of entry.

Conclusion:

As shown in FIG. 112, single PE (No. 38) could effectively deliver sRNAsingle-stranded nucleic acid into mouse blood via oral administration,and the delivery effect was better than POPC and Lipofectamine RNAiMAX.

Example 12-3: Delivery of Single-Stranded Nucleic Acids by Single LipidNo. 40 In Vivo

1. Experimental animals: C57 mice, male, approximately 6 weeks old.

1) Naive group: intragastric administration of 500 μl saline;

2) RNAiMAX treatment group: 10 μl RNAiMAX-1 nmol sRNA was mixed andintragastrically administered to each mouse. This group served as apositive control group. RNAiMAX was purchased from Invitrogen.

3) Free uptake group: single-stranded sRNA mixture solution was directlyadded (each 1 nmol);

4) Treatment group of POPC and nucleic acid: a mixture of 10 μL POPC andsingle-stranded sRNA (each 1 nmol) mixture solution that was treated byheating method was given to mice by gavage.

5) Treatment group of single lipid and nucleic acid mixture: a mixtureof a 10 μL single lipid (No. 40) and single-stranded sRNA (PGY-sRNA-32and PGY-sRNA-26, 1 nmol each) mixture solution that was treated byheating method was given to mice by gavage.

2. 12 hours after gavage, the blood was taken from the eyeball and lysedby TRIzol to extract RNA for the detection of the amount of entry.

Conclusion:

As shown in FIG. 113, single PE (No. 40) could effectively deliver sRNAsingle-stranded nucleic acid into mouse blood via oral administration,and the delivery effect was better than POPC and Lipofectamine RNAiMAX.

Example 12-4: Delivery of Single-Stranded Nucleic Acids by Single LipidNo. 64 In Vivo

1. Experimental animals: C57 mice, male, approximately 6 weeks old.

1) Naive group: intragastric administration of 500 μl saline;

2) RNAiMAX treatment group: 10 μl RNAiMAX-1 nmol sRNA was mixed andintragastrically administered to each mouse. This group served as apositive control group. RNAiMAX was purchased from Invitrogen.

3) Free uptake group: single-stranded sRNA mixture solution was directlyadded (each 1 nmol);

4) Treatment group of POPC and nucleic acid: a mixture of 10 μL POPC andsingle-stranded sRNA (each 1 nmol) mixture solution that was treated byheating method was given to mice by gavage.

5) Treatment group of single lipid and nucleic acid mixture: a mixtureof a 10 μL single lipid (No. 64) and single-stranded sRNA (PGY-sRNA-32,1 nmol each) mixture solution that was treated by heating method wasgiven to mice by gavage.

2. 12 hours after gavage, the blood was taken from the eyeball and lysedby TRIzol to extract RNA for the detection of the amount of entry.

Conclusion:

As shown in FIG. 114, single PE (No. 64) could effectively deliver sRNAsingle-stranded nucleic acid into mouse blood via oral administration,and the delivery effect was better than POPC and Lipofectamine RNAiMAX.

Example 12-5: Delivery of Single-Stranded Nucleic Acids by Single LipidNo. 71 In Vivo

1. Experimental animals: C57 mice, male, approximately 6 weeks old.

1) Naive group: intragastric administration of 500 μl saline;

2) RNAiMAX treatment group: 10 μl RNAiMAX-1 nmol sRNA was mixed andintragastrically administered to each mouse. This group served as apositive control group. RNAiMAX was purchased from Invitrogen.

3) Free uptake group: single-stranded sRNA mixture solution was directlyadded (each 1 nmol);

4) Treatment group of POPC and nucleic acid: a mixture of 10 μL POPC andsingle-stranded sRNA (each 1 nmol) mixture solution that was treated byheating method was given to mice by gavage.

5) Treatment group of single lipid and nucleic acid mixture: a mixtureof a 10 μL single lipid (No. 71) and single-stranded sRNA mixture(PGY-sRNA-32, 1 nmol each) solution that was treated by heating methodwas given to mice by gavage.

2. 12 hours after gavage, the blood was taken from the eyeball and lysedby TRIzol to extract RNA for the detection of the amount of entry.

Conclusion:

As shown in FIG. 115, single PE (No. 71) could effectively deliver sRNAsingle-stranded nucleic acid into mouse blood via oral administration,and the delivery effect was better than POPC and Lipofectamine RNAiMAX.

Example 13: Lipids Effectively Deliver Single-Stranded Nucleic Acidsinto MRC-5 Cell at Different Temperature Gradients

1. Experimental Groups:

1) Naive group: untreated cells;

2) RNAiMAX treatment group: 2 μL RNAiMAX transfection reagent andsingle-stranded HJT-sRNA-m7 solution were diluted in 100 μL opti-MEMmedium respectively, and then the two were mixed, allowed to stay for 15min, added into the cells, and then mixed, and the final concentrationof single-stranded HJT-sRNA-m7 was 100 nM;

3) Treatment group of single lipid and nucleic acid mixture: mixtures of2.5 μL, single lipid (No. 38) and HJT-sRNA-m7 double-stranded nucleicacid solution that were treated by boiling method at differenttemperatures was added to the cells and then mixed, and the finalconcentration of RNA was 100 nM.

4° C.: to 100 μL single-stranded HJT-sRNA-m7 solution was added 2.5 μLsingle lipid and placed at 4° C. for 15 min; 6 hours after the additionof the cells, the expression level of HJT-sRNA-m7 in cells was detectedby RT-qPCR.

37° C.: to 100 μL single-stranded HJT-sRNA-m7 solution was added 2.5 μLsingle lipid and placed at 37° C. for 15 min. 6 hours after the additionof the cells, the expression level of HJT-sRNA-m7 in cells was detectedby RT-qPCR.

60° C.: to 100 μL single-stranded HJT-sRNA-m7 solution was added 2.5 μLsingle lipid and heated at 60° C. for 15 min. 6 hours after the additionof the cells, the expression level of HJT-sRNA-m7 in cells was detectedby RT-qPCR.

80° C.: to 100 μL single-stranded HJT-sRNA-m7 solution was added 2.5 μLsingle lipid and heated at 80° C. for 15 min. 6 hours after the additionof the cells, the expression level of HJT-sRNA-m7 in cells was detectedby RT-qPCR.

100° C.: to 100 μL HJT-sRNA-m7 single-stranded solution was added 2.5 μLsingle lipid and heated at 100° C. for 15 min. 6 hours after theaddition of the cells, the expression level of HJT-sRNA-m7 in cells wasdetected by RT-qPCR.

Conclusion:

As shown in FIG. 116, results showed that the lipids by the boilingmethod at different temperate conditions could effectively delivernucleic acids into cells (statistically significant, p<0.01), having thepotential of improving the efficiency of the delivery of nucleic aciddrug in clinical settings.

1-66. (canceled)
 67. A method of delivering a nucleic acid using a lipidcomposition, comprising mixing the lipid composition with the nucleicacid to obtain a lipid nucleic acid mixture, and delivering the lipidnucleic acid mixture so that the nucleic acid is delivered, wherein thelipid composition comprising one or more compounds having the followingformula:

wherein L₁, L₂, and L₃ are each independently selected from the groupconsisting of null, —C(O)O—CH₂—, —CH(OH)—, —C(O)—NH—CH₂—, —CH₂—OC(O)—,—CH₂—NH—C(O)—, —C(O)O—, —C(O)NH—, —OC(O)—, —NH—C(O)—, —CH₂—,

with the proviso that at most two of L₁, L₂ and L₃ are null; withrespect to the divalent groups L₁ and L₂, the dash “-” on the left sideis linked to the groups A and B, respectively, and the dash “-” on theright side is linked to the central carbon atom; with respect to thedivalent group L₃, the dash “-” on the left side is linked to thecentral carbon atom, and the dash “-” on the right side is linked to thegroup Q; A, B and Q are each independently selected from the groupconsisting of H, —OH, C₁₋₂₀ alkyl, C₁₋₂₀ alkenyl, C₁₋₂₀ heteroalkyl,C₁₋₂₀ heteroalkenyl, —NH₂, and —NR₃ ⁺, R is each independently H or C₁₋₆alkyl; and n is an integer 0, 1, 2, 3 or 4;
 68. The method of claim 67,wherein the nucleic acid is delivered by oral administration, inhalationor injection.
 69. The method of claim 67, wherein the nucleic acid isdelivered to a target cell.
 70. The method of claim 67, wherein thenucleic acid is delivered to a subject in need thereof.
 71. The methodof claim 70, wherein the nucleic acid treats a disease in the subject.72. The method of the claim 71, wherein the disease is a cancer.
 73. Themethod of claim 67, wherein lipid nucleic acid is obtained by a boilingmethod, a reverse evaporation method, or by direct mixing.
 74. Themethod of claim 73, wherein the temperature of the boiling method beingselected from group consisting of a range of 25° C. to 100° C., a rangeof 50° C. to 100° C., a range of 80° C. to 100° C., and a range of 95°C. to 100° C.
 75. The method of claim 73, wherein the boiling methodcomprises adding an organic solvent solution of the lipid composition toan aqueous solution of the nucleic acid.
 76. The method of claim 73,wherein the temperature of the reverse evaporation method is 25° C. to70° C., 30° C. to 65° C., or 40° C. to 60° C.
 77. The method of claim73, wherein the reverse evaporation method comprises adding an aqueoussolution of the nucleic acid to an organic solvent solution of the lipidcomposition.
 78. The method of claim 67, wherein L₁ is selected from thegroup consisting of null, —C(O)O—CH₂— and —CH(OH)—, L₂ is selected fromthe group consisting of null, —C(O)O— and —C(O)NH—, L₃ is selected fromthe group consisting of null, —C(O)O—, —CH₂—OC(O)—, —CH₂— and

A is selected from the group consisting of H, —OH, C₁₋₂₀ alkyl and C₁₋₂₀alkenyl; B is selected from the group consisting of H, —OH, —NH₂, C₁₋₂₀alkyl and C₁₋₂₀ alkenyl; Q is selected from the group consisting of H,—OH, C₁₋₂₀ alkyl and C₁₋₂₀ alkenyl, —NH₂, and —NR₃ ⁺, wherein R is H orC₁₋₆ alkyl.
 79. The method of claim 78, wherein the compound is selectedfrom the group consisting of:

wherein A is selected from the group consisting of a linear C₁₅₋₁₈ alkylgroup and a linear C₁₅₋₁₈ alkenyl group; B is selected from the groupconsisting of a linear C₁₅₋₁₈ alkyl group and a linear C₁₅₋₁₈ alkenylgroup; Q is selected from the group consisting of H, —OH, a linearC₁₅₋₁₈ alkyl group and a linear C₁₅₋₁₈ alkenyl group, and —NR₃ ⁺,wherein R is H or methyl; and L₃ is —C(O)O—.
 80. The method of claim 67,wherein the lipid composition comprises any one or more lipids selectedfrom Table 1, or a combination of the any one or more lipids selectedfrom Table 1 with any one or more other lipids and other relatedchemicals.
 81. The method of claim 80, wherein the lipid compositioncomprises any one or more lipids selected from the following group, or acombination thereof with any one or more other lipids and other relatedchemicals:


82. The method of claim 67, wherein the nucleic acid is a small nucleicacid.
 83. The method of claim 67, wherein the nucleic acid is singlestranded or double stranded.
 84. The method of claim 67, wherein thenucleic acid has a stem-loop structure.
 85. The method of claim 67,wherein the nucleic acid has a length of 14-32 bp, 16-28 bp or 18-24 bp.86. A compound having the following structure:


87. A pharmaceutical composition, comprising a lipid composition and anucleic acid, wherein the lipid composition comprises one or morecompounds having the following formula:

wherein L₁, L₂, and L₃ are each independently selected from the groupconsisting of null, —C(O)O—CH₂—, —CH(OH)—, —C(O)—NH—CH₂—, —CH₂—OC(O)—,—CH₂—NH—C(O)—, —C(O)O—, —C(O)NH—, —OC(O)—, —NH—C(O)—, —CH₂—,

with the proviso that at most two of L₁, L₂ and L₃ are null; A, B and Qare each independently selected from the group consisting of H, —OH,C₁₋₂₀ alkyl, C₁₋₂₀ alkenyl, C₁₋₂₀ heteroalkyl, C₁₋₂₀ heteroalkenyl,—NH₂, and —NR₃ ⁺, R is H or C₁₋₆ alkyl; and n is an integer 0, 1, 2, 3or
 4. 88. The pharmaceutical composition of claim 87, wherein thepharmaceutical composition is formulated so that it can be administeredvia oral administration, inhalation, digestive tract or respiratorytract.
 89. The pharmaceutical composition of claim 87, wherein at leastpart of or all of the lipid composition and the nucleic acid exist inthe form of a mixture.
 90. The pharmaceutical composition of claim 87,wherein the pharmaceutical composition is provided in the form of a kit,the lipid composition and the nucleic acid in the kit being eachindependently provided in a first container and a second container, thefirst container and the second container being the same or different.91. A method for the manufacture of a pharmaceutical composition,comprising adding an organic solvent solution comprising a lipid to anaqueous solution of a nucleic acid, and subsequently heating at atemperature selected from the group consisting of a range of 25° C. to100° C., a range of 50° C. to 100° C., a range of 80° C. to 100° C., anda range of 95° C. to 100° C. to obtain the pharmaceutical composition.92. A lipid composition selected from any one of the following: a lipidcombination of No. 8:No. 41=6:1; a lipid combination of No. 38:No.41=6:1; a lipid combination of No. 39:No. 41=6:1; a lipid combination ofNo. 40:No. 41=6:1; a lipid combination of No. 38:No. 12:No. 41:No.29=1:1:2:1; a lipid combination of No. 40:No. 12:No. 41=2:4:3; a lipidcombination of No. 12:No. 41=1:6; a lipid combination of No. 12:No.41=1:1; a lipid combination of No. 12:No. 41=6:1; a lipid combination ofNo. 40:No. 12:No. 41=2:2:2; a lipid combination of No. 4:No. 12:No.41=1:1:1; DG combination of No. 1:No. 2:No. 3:No. 19:No. 35=1:1:1:1:1;TG combination of No. 6:No. 9:No. 10:No. 13:No. 15:No. 16:No. 18:No.20:No. 21:No. 22:No. 23:No. 24:No. 25:No. 26:No. 27:No. 28:No. 32:No.33=1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1; LPC combination of No. 36:No.37=1:1; PC combination of No. 11:No. 12=1:1; PE combination of No. 8:No.38=1:1; Cer combination of No. 4:No. 14=1:1; So combination of No.17:No. 30:No. 31=1:1:1; an equal volume combination of No. 1-36 withoutNo. 5, No. 7; an equal volume combination of No. 1-36 without No. 5, No.7, No. 34; an equal volume combination of No. 1-36 without No. 5, No. 7,No. 1, No. 2, No. 3, No. 19, No. 35; an equal volume combination of No.1-36 No. 5, No. 7, No. 6, No. 9, No. 10, No. 13, No. 15, No. 16, No. 18,No. 20, No. 21, No. 22, No. 23, No. 24, No. 25, No. 26, No. 27, No. 28,No. 32, No. 33; an equal volume combination of No. 1-36 without No. 5,No. 7, No. 36, No. 37; an equal volume combination of No. 1-36 withoutNo. 5, No. 7, No. 11, No. 12; an equal volume combination of No. 1-36without No. 5, No. 7, No. 8 in; an equal volume combination of No. 1-36without No. 5, No. 7, No. 4, No. 14; an equal volume combination of No.1-36 without No. 5, No. 7, No. 29; a lipid combination of No. 1:No.34=2:1; a lipid combination of No. 1: the DG composition=2:1; a lipidcombination of No. 1: the TG composition=2:1; a lipid combination of No.1: the LPC composition=2:1; a lipid combination of No. 1:No. 8=2:1; alipid combination of No. 1:No. 12=2:1; a lipid combination of No. 1: theCer composition=2:1; a lipid combination of No. 1: the Socomposition=2:1; a lipid combination of No. 1:No. 29=2:1; a lipidcombination of No. 1:No. 8:No. 12=1:1:1; a lipid combination of No.8:No. 34=2:1; a lipid combination of No. 8: the DG composition=2:1; alipid combination of No. 8: the TG composition=2:1; a lipid combinationof No. 8: the LPC composition=2:1; a lipid combination of No. 8:No.37=4:1; a lipid combination of No. 8:No. 12=2:1; a lipid combination ofNo. 8: the Cer composition=2:1; a lipid combination of No. 8: the Socomposition=2:1; a lipid combination of No. 8:No. 31=6:1; a lipidcombination of No. 8:No. 29=2:1; a lipid combination of No. 12:No.34=2:1; a lipid combination of No. 12: the DG composition=2:1; a lipidcombination of No. 12: the TG composition=2:1; a lipid combination ofNo. 12: the LPC composition=2:1; a lipid combination of No. 12:No.8=2:1; a lipid combination of No. 12: the Cer composition=2:1; a lipidcombination of No. 12: the So composition=2:1; a lipid combination ofNo. 12:No. 29=2:1; a lipid combination of No. 12:No. 8:No. 1&2=2:1:1; alipid combination of No. 12:No. 8:No. 15=2:1:1; a lipid combination ofNo. 12:No. 8:No. 36&37=2:1:1; a lipid combination of No. 12:No. 8:No.11=2:1:1; a lipid combination of No. 12:No. 8:No. 12=2:1:1; a lipidcombination of No. 12:No. 8:No. 4=2:1:1; a lipid combination of No.12:No. 8:No. 31=2:1:1; a lipid combination of No. 12:No. 8:No. 29=2:1:1;a lipid combination of No. 12:No. 8:No. 34=3:2:1; a lipid combination ofNo. 12:No. 8:No. 34=4:2:3; a lipid combination of No. 12:No. 8:No.2=4:2:3; a lipid combination of No. 12:No. 8:No. 2=16:8:3; a lipidcombination of No. 12:No. 8:No. 32=4:2:3; a lipid combination of No.12:No. 8:No. 37=4:2:3; a lipid combination of No. 12:No. 8:No. 11=4:2:3;a lipid combination of No. 12:No. 8:No. 38=4:2:3; a lipid combination ofNo. 12:No. 8:No. 4=4:2:3; a lipid combination of No. 12:No. 8:No.31=4:2:3; a lipid combination of No. 12:No. 8:No. 29=4:2:3; a lipidcombination of No. 12:No. 8:No. 29:No. 31=2:1:1:1; a lipid combinationof No. 12:No. 8:No. 29:No. 31:No. 34=4:2:2:2:5; a lipid combination ofNo. 12:No. 8:No. 29:No. 31:No. 2=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 32=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 11=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 37=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 38=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 4=4:2:2:2:5; a lipid combination of No.12:No. 8:No. 29:No. 31:No. 4:No. 1:No. 16=2:1:1:3:2:2:3; a lipidcombination of No. 1:No. 8:No. 12:No. 1&2=2:2:2:3; a lipid combinationof No. 1:No. 8:No. 12:No. 15=2:2:2:3; a lipid combination of No. 1:No.8:No. 12:No. 36&37=2:2:2:3; a lipid combination of No. 1:No. 8:No.12:No. 12=2:2:2:3; a lipid combination of No. 1:No. 8:No. 12:No.4=2:2:2:3; a lipid combination of No. 1:No. 8:No. 12:No. 31=2:2:2:3; alipid combination of No. 1:No. 8:No. 12:No. 29=2:2:2:3; a lipidcombination of No. 8:No. 34:No. 1&2=2:1:1; a lipid combination of No.8:No. 34:No. 15=2:1:1; a lipid combination of No. 8:No. 34:No.36&37=2:1:1; a lipid combination of No. 8:No. 34:No. 12=2:1:1; a lipidcombination of No. 8:No. 34:No. 4=2:1:1; a lipid combination of No.8:No. 34:No. 31=2:1:1; a lipid combination of No. 8:No. 34:No. 29=2:1:1;a lipid combination of No. 8:No. 31:No. 34=12:3:5; a lipid combinationof No. 8:No. 31:No. 2=12:3:5; a lipid combination of No. 8:No. 31:No.37=12:3:5; a lipid combination of No. 8:No. 31:No. 11=12:3:5; a lipidcombination of No. 8:No. 31:No. 12=12:3:5; a lipid combination of No.8:No. 31:No. 4=12:3:5; a lipid combination of No. 8:No. 31:No.29=12:3:5; a lipid combination of No. 8:No. 31:No. 32=12:3:5; a lipidcombination of No. 8:No. 4:No. 34=12:3:5; a lipid combination of No.8:No. 4:No. 2=12:3:5; a lipid combination of No 0.8:No. 4:No. 37=12:3:5;a lipid combination of No. 8:No. 4:No. 12=12:3:5; a lipid combination ofNo. 8:No. 4:No. 31=12:3:5; a lipid combination of No. 8:No. 4:No.29=12:3:5; a lipid combination of No. 8:No. 4:No. 32=12:3:5; a lipidcombination of No. 38:No. 34=2:1; a lipid combination of No. 38:No.1=2:1; a lipid combination of No. 38:No. 2=2:1; a lipid combination ofNo. 38:No. 1&2=2:1; a lipid combination of No. 38:No. 15=2:1; a lipidcombination of No. 38:No. 32=2:1; a lipid combination of No. 38:No.37=2:1; a lipid combination of No. 38:No. 37=4:1; a lipid combination ofNo. 38:No. 11=2:1; a lipid combination of No. 38:No. 12=2:1; a lipidcombination of No. 38:No. 11&12=2:1; a lipid combination of No. 38:No.12=4:1; a lipid combination of No. 38:No. 8=2:1; a lipid combination ofNo. 38:No. 4=2:1; a lipid combination of No. 38:No. 30=2:1; a lipidcombination of No. 38:No. 31=2:1; a lipid combination of No. 38:No.29=2:1; a lipid combination of No. 1:No. 38:No. 12:No. 34=2:2:2:3; alipid combination of No. 1:No. 38:No. 12:No. 15=2:2:2:3; a lipidcombination of No. 1:No. 38:No. 12:No. 37=2:2:2:3; a lipid combinationof No. 1:No. 38:No. 12:No. 8=2:2:2:3; a lipid combination of No. 1:No.38:No. 12:No. 4=2:2:2:3; a lipid combination of No. 1:No. 38:No. 12:No.31=2:2:2:3; a lipid combination of No. 1:No. 38:No. 12:No. 29=2:2:2:3; alipid combination of No. 38:No. 34:No. 1=2:1:3; a lipid combination ofNo. 38:No. 34:No. 15=2:1:3; a lipid combination of No. 38:No. 34:No.37=2:1:3; a lipid combination of No. 38:No. 34:No. 12=2:1:3; a lipidcombination of No. 38:No. 34:No. 8=2:1:3; a lipid combination of No.38:No. 34:No. 4=2:1:3; a lipid combination of No. 38:No. 34:No.31=2:1:3; a lipid combination of No. 38:No. 34:No. 29=2:1:3; a lipidcombination of No. 38:No. 12:No. 1=2:1:3; a lipid combination of No.38:No. 12:No. 2=4:1:3; a lipid combination of No. 38:No. 12:No.15=2:1:3; a lipid combination of No. 38:No. 12:No. 37=2:1:3; a lipidcombination of No. 38:No. 12:No. 8=2:1:3; a lipid combination of No.38:No. 12:No. 4=2:1:3; a lipid combination of No. 38:No. 12:No.31=2:1:3; a lipid combination of No. 38:No. 12:No. 29=2:1:3; a lipidcombination of No. 38:No. 12:No. 1:No. 15:No. 34=22:22:22:33:36; a lipidcombination of No. 38:No. 12:No. 1:No. 15:No. 37=22:22:22:33:36; a lipidcombination of No. 38:No. 12:No. 1:No. 15:No. 4=22:22:22:33:36; a lipidcombination of No. 38:No. 12:No. 1:No. 15:No. 31=22:22:22:33:36; a lipidcombination of No. 38:No. 12:No. 1:No. 15:No. 29=22:22:22:33:36; a lipidcombination of No. 38:No. 34:No. 37:No. 1=44:22:33:36; a lipidcombination of No. 38:No. 34:No. 37:No. 15=44:22:33:36; a lipidcombination of No. 38:No. 34:No. 37:No. 12=44:22:33:36; a lipidcombination of No. 38:No. 34:No. 37:No. 4=44:22:33:36; a lipidcombination of No. 38:No. 34:No. 37:No. 31=44:22:33:36; a lipidcombination of No. 38:No. 12:No. 4:No. 34=44:22:33:36; a lipidcombination of No. 38:No. 12:No. 4:No. 1=44:22:33:36; a lipidcombination of No. 38:No. 12:No. 4:No. 15=44:22:33:36; a lipidcombination of No. 38:No. 12:No. 4:No. 37=44:22:33:36; a lipidcombination of No. 38:No. 12:No. 4:No. 37=8:2:5:3; a lipid combinationof No. 38:No. 12:No. 4:No. 31=44:22:33:36; a lipid combination of No.38:No. 12:No. 4:No. 29=44:22:33:36; a lipid combination of No. 38:No.12:No. 4:No. 29:No. 34=88:44:66:72:135; a lipid combination of No.38:No. 12:No. 4:No. 29:No. 1=88:44:66:72:135; a lipid combination of No.38:No. 12:No. 4:No. 29:No. 15=88:44:66:72:135; a lipid combination ofNo. 38:No. 12:No. 4:No. 29:No. 37=88:44:66:72:135; a lipid combinationof No. 38:No. 12:No. 4:No. 29:No. 31=88:44:66:72:135; a lipidcombination of No. 38:No. 12:No. 4:No. 2=20:10:15:9; a lipid combinationof No. 38:No. 12:No. 4:No. 6=20:10:15:9; a lipid combination of No.38:No. 12:No. 4:No. 17=20:10:15:9; a lipid combination of No. 38:No.12:No. 4:No. 29=20:10:15:9; a lipid combination of No. 38:No. 12:No.4:No. 34=20:10:15:9; a lipid combination of No. 38:No. 12:No. 4:No.37=20:10:15:9; a lipid combination of No. 38:No. 12:No. 31:No.34=2:1:3:3; a lipid combination of No. 38:No. 12:No. 31:No. 1=2:1:3:3; alipid combination of No. 38:No. 12:No. 31:No. 15=2:1:3:3; a lipidcombination of No. 38:No. 12:No. 31:No. 37=2:1:3:3; a lipid combinationof No. 38:No. 12:No. 31:No. 4=2:1:3:3; a lipid combination of No. 38:No.12:No. 31:No. 29=2:1:3:3; a lipid combination of No. 38:No. 34:No.37:No. 31:No. 1=88:44:66:72:135; a lipid combination of No. 38:No.34:No. 37:No. 31:No. 15=88:44:66:72:135; a lipid combination of No.38:No. 34:No. 37:No. 31:No. 12=88:44:66:72:135; a lipid combination ofNo. 38:No. 34:No. 37:No. 31:No. 4=88:44:66:72:135; a lipid combinationof No. 38:No. 34:No. 37:No. 31:No. 29=88:44:66:72:135; a lipidcombination of No. 38:No. 37:No. 34=4:2:3; a lipid combination of No.38:No. 37:No. 1=4:2:3; a lipid combination of No. 38:No. 37:No. 2=4:2:3;a lipid combination of No. 38:No. 37:No. 1&2=4:2:3; a lipid combinationof No. 38:No. 37:No. 2=32:8:5; a lipid combination of No. 38:No. 37:No.32=32:8:5; a lipid combination of No. 38:No. 37:No. 15=4:2:3; a lipidcombination of No. 38:No. 37:No. 32=4:2:3; a lipid combination of No.38:No. 37:No. 8=4:2:3; a lipid combination of No. 38:No. 37:No.11=4:2:3; a lipid combination of No. 38:No. 37:No. 12=4:2:3; a lipidcombination of No. 38:No. 37:No. 11&12=4:2:3; a lipid combination of No.38:No. 37:No. 12=4:1:1; a lipid combination of No. 38:No. 37:No.4=4:2:3; a lipid combination of No. 38:No. 37:No. 30=4:2:3; a lipidcombination of No. 38:No. 37:No. 31=4:2:3; a lipid combination of No.38:No. 37:No. 29=4:2:3; a lipid combination of No. 8:No. 37:No.32=4:1:2; a lipid combination of No. 8:No. 37:No. 2=4:1:2; a lipidcombination of No. 38:No. 37:No. 15:No. 34=64:16:10:45; a lipidcombination of No. 38:No. 37:No. 15:No. 1=64:16:10:45; a lipidcombination of No. 38:No. 37:No. 15:No. 12=64:16:10:45; a lipidcombination of No. 38:No. 37:No. 15:No. 4=64:16:10:45; a lipidcombination of No. 38:No. 37:No. 15:No. 31=64:16:10:45; a lipidcombination of No. 38:No. 37:No. 15:No. 29=64:16:10:45; a lipidcombination of No. 38:No. 2:No. 37=4:2:3; a lipid combination of No.38:No. 2:No. 31=4:2:3; a lipid combination of No. 38:No. 2:No. 29=4:2:3;a lipid combination of No. 38:No. 2:No. 34=4:2:3; a lipid combination ofNo. 38:No. 2:No. 32=4:2:3; a lipid combination of No. 38:No. 2:No.12=4:2:3; a lipid combination of No. 38:No. 2:No. 12=4:5:1; a lipidcombination of No. 38:No. 2:No. 4=4:2:3; No. 1&2, No. 11&12 and No.36&37 represent lipids No. 1 and No. 2 in any ratio, lipids No. 11 andNo. 12 in any ratio, lipids No. 36 and No. 37 in any ratio,respectively.